Lemongrab and his clone are all but outright stated to be a couple, complete with Headbutt of Love within seconds of meeting each other, and Bubblegum saying "I made him, for you to be with". Then "All Your Fault" goes into full Adam and Eve Plot, with the two starting a family together (and bickering with each other over what to name their children). However, they at least once refer to each other as "brother."
Princess Bubblegum showed no signs of returning Finn's three-season-long Precocious Crush, and didn't have any interest in any of the suitors (who were all male) in "The Suitor". Meanwhile, "What Was Missing" heavily implies she had a much closer relationship with Marceline and still yearns to have it again, implications further explored in "Sky Witch"; in the beginning of the episode she is seen heavily sniffing the shirt that Marceline gave to her (which was stated in "What Was Missing" as her most prized possession), then towards the end the episode we discover that she used her shirt to make a trade with Maja the Sky Witch to get Hambo back to Marceline, because of all the emotions it contains. After years of speculation, Marceline's voice actress Olivia Olsen finally confirmed that she was told by the writers that Bubblegum and Marceline used to date, removing the "ambiguous" status. Though she mentionned in Slumber Party that she used to date Mr. Cream Puff.
Everything about Abracadaniel is soft and delicate, his speech included, he uses rainbow magic, is outright repulsed at the idea of kissing Bubblegum in "Wizard Battle", gets very chummy with Ice King very quickly in "Play Date", and is shaped like a giant pink penis.
Parodied with the cartoons as superheroes Ace and Gary fight crime with an array of phallically-suggestive vehicles and gadgets while engaging in plenty of Ho Yay behavior, all the while their villains dance around the obviousness of their gay-ness.
On the commentary for the "Best of TV Funhouse" DVD, SNL writer Robert Smigel said "one of them is gay, and one of them is straight, which will lead to a breakup". He refused to say which was which.
Mr. Ratburn is single, thin and neat. With the exception of some earlier episodes, he speaks in a somewhat "sing-songy" way compared to other adult male characters. His hobby is performing puppet shows, and he owns and maintains quite a collection of marionettes.
Binky's Hidden Depths reveal an interest for stereotypically gay hobbies such as theater, dance (especially ballet), art, music, culture, and the likes.
Brandon Higsby. He is rather girly looking, is extremely hyper, is always neatly dressed and mentioned in one episode that he dreams about purple unicorns and he likes the color pink and teddy bear pencils.
Blake Gripling always seemed a little too obsessed with winning Carl's friendship and hates Carl's best friend Hoodsey.
Courtney, although very girly, gives off these vibes near-constantly, especially when interacting with Ginger. She is attracted to boys several times however the fanon consensus is she's probably gay and doesn't know it or represses it.
In the Batman: The Animated Series episode "Feat of Clay", Matt Hagen (who becomes Clayface) has a partner and stand-in called Teddy Lupin, who shows some very ambiguous signs. At first glance they might just be good friends, but with Teddy grabbing Matt's arm out of joy, mothering and looking after him constantly, and saying things such as "we can go on!", not to mention Hagen's violent mood swings, it's hard not to interpret them as an abusive couple. Hagen, however, shows affection for a female character in a later episode "Mudslide", suggesting that he is either bisexual, or that the feeling was not mutual, or that there was no relationship whatsoever, or that he's happy with using and abusing anyone who happens to fall in love with him, regardless of gender.
In the banned Dexter's Laboratory episode "Barbequor", the title character's sidekick The Silver Spooner (a Silver Surfer parody) was extremely effeminate and campy, including the limp wrist, he skipped when excited and raised one leg, walking on his tiptoes while swinging his hips, and slapping Monkey to prevent him from getting to his master.
The entire cast, especially in regards to the bizarrely pervertedEnsemble Dark Horse Captain Hero and Xandir P. Wifflebottom who stated "I'm on a never ending quest to save my girlfriend!" for the first three episodes, until he finally faces his sexuality and then changes that to, "I'm on a never ending quest to save my boyfriend!"
There's also Wooldoor Sockbat and Spanky Ham. When Captain Hero simulates sexual advances to Wooldoor, he swoons and says, "Captain Hero? I want you inside me." To which he responds, "Class dismiiiised." As for Spanky, he was right in the middle of a triple French kiss with Hero and Wooldor. Not so ironically, the ladies took "poker night" surprisingly well.
The episode where Xandir came out also featured other ambiguously gay characters such as Elmer Fudd and Snagglepuss.
Droids: The Adventures of R2-D2 and C-3PO: C-3PO became a gay icon shortly after his film debut, but the 1985 spinoff cartoon Droids takes the implications Up to Eleven. In addition to being mincing and prissy, Threepio has limp wrists, seems legitimately uncomfortable when a woman hugs him, harbors a fondness for fashion accessories, and shares a surprising amount of Ho Yay with R2-D2.
Cupid, such as in the "Wishology" trilogy where Cupid and Juandissimo had a scene in "The Exciting Middle Part" eating in an outdoor cafe together, acting very much like a dating couple (which would make Juandissimo Ambiguously Bi.
Chester doesn't like being too close to girls because he gets rashes, an episode had Chester cross-dressing and dancing with AJ when the two needed dance partners.
George And Martha: Oscar & Wilde. Apparently in the short lived cartoon series, there is a pair of alligator characters who give off so many clear signs that there is definitely something more to their relationship. They live together, they go on vacation together, one is never seen without the other, and there's even a scene in one episode where the two are dressed up in outfits straight out of the Village People. The kicker? Their names are Oscar and Wilde. It's like they were trying to see how far they could go in a children's cartoon without outright stating the fact.
Gravity Falls: Sheriff Blubs and Deputy Durland tend to act like a married couple. Thankfully though, any humor the show aims at them isn't because of this trope, but because they're morons.
There's also Tyler the Cute Biker. No man in Gravity Falls wears shorts as short as his, and he was also seen at the Sev'ral Timez concert, so.....
Samy Garvin, who can best be described as Smithers from the Simpsons in demon form. The fact that he has Always Camp dreams of stardom do not help his case.
The titular Jimmy Two-Shoes as well. He has considerable Ho Yay towards Lucius and in Matchmaker Jimmy, when Lucius tells Beezy he needs a date for an upcoming event, Beezy turns to Jimmy and asks him how he looks in a dress. Jimmy responds with "What kind of a dress?"
Johnny Bravo: Carl pounces on Johnny constantly (as in "Johnny Goes to Camp"), is very clingy despite Johnny shoving him away, all-too-into playing Johnny's pretend date ("Charm School Johnny") or wife ("Chain Gang Johnny"), is impervious to the wiles of chicks, often thinking they're evil ("Forest Chump"), and even said he loved Johnny while standing up for him against an angry mob in "Lodge Brother Johnny". Heck, the episode "Carl Be Not Proud" is practically nothing BUT Ho Yay.
Dukey, his trademark laugh, some of the outfits he wears (particularly the vampire hunter episode) and getting cuddly with Johnny in the episode Johnny Irresistable to the point where he nearly starts humping Johnny's leg.
Legion of Super Heroes has Color Kid, who wears a rainbow on his chest, as well as speaking and gesturing flamboyantly. He's also concerned at one point that he may not have used the precisely appropriate shade of green.
Gilda acts like a jealous girlfriend to Rainbow Dash in "Griffon the Brush Off", bordering on Psycho Ex-Girlfriend, but no romantic feelings are ever mentioned. Which only added to the already-rampant speculation about Rainbow Dash's tomboy personality and connection with rainbows.
Stephen Magnet, the Sea Serpent from part 2 of the pilot. He's pinkish-purple, speaks in an effeminate voice, and cries when his fabulous mustache is ruined.
Every single pony from G1 My Little Pony, though a few stand out. Applejack licked Glory in the beginning of the first special, though it seems like it's just affection and due to the fact she's covered in apple juice - it seems innocent until you notice some official figurines have Glory marrying Moondancer, another mare. There's also Gusty, who fits for most of the same reason Dash does in G4 (plus she has an androgynous voice).
Vice-Principal Zeigler from Pelswick has a lot of stereotypical mannerisms, was just as excited if not more than the middle-school girls to see the boy band, N*Talented (whom all the other guys at least tried to hate), and occasionally dresses in a fairy costume. In one episode, Ace voices suspicions that Pelswick's online girlfriend might actually be a guy, and the hypothetical scenario involved the guy in question being him in the fairy costume. In another, Pelswick's grandmother refers to him as a "fruitcake."
King Julien. As mentioned in the film above, but is even more noticeable here, although he shows interest in courting the only main female character, Marlene the otter, and has also admitted to having girlfriends in Canada (no, really). Some of his dialogue can easily be interpreted as a ring-tailed lemur in serious denial. (Six words: "No one will be sucking seed!")
Mort might be a close second, though he's only interested in Julien's feet specifically, not the rest of him.
Roger the alligator, who doesn't want to scare anyone; he wants to bake muffins for bullies, and wants to sing on Broadway.
All the penguins have their moments, but those made the biggest deal of are Private and Rico - Private could be acting babyish, but it's just funny for the Dumb Muscle to have quite the number of notable 'gay' moments.
The Problem Solverz has Roba, the most sensitive member of the team and is frequently anxious about being loved and accepted. He practically had a man crush on master funny face artist Tony Marv, and becomes upset in another episode because he wouldn't be able to wear a salsa hat if Bad Cat went undefeated. Roba was also completely turned off by Katrina Rad's amorous advances (although she wascrazy), and is quick is criticize Alfe's fashion sense. His high-pitched voice and effeminate hand gestures are additional traits.
The Proud Family: Michael the "sissy" sole male cheerleader and son of the macho coach(who he can't call "Dad" in public).
The Reluctant Dragon: In the Disney animated short, the title character is rather effeminate and very campy, singing in a high voice, reciting poetry, dancing on his tiptoes, doing the stereotypical gay wave, etc.
The title characters of The Ren & Stimpy Show, whose ever ambiguous status as a couple was something of a Running Gag throughout the series. When asked about fan rumors that they were a gay couple their creator reportedly just gave a Shrug of God and said "I don't know, it's none of my business". In the Adult Party Cartoon, however, it was made... quite explicit... that the duo were in fact lovers. This kind of ruined the joke, though.
Mr. Waylon Smithers used to be Ambiguously Gay but in later seasons the show just ran with the gay jokes. Originally he was strictly a "Burnsosexual", which eventually devolved into Ambiguously Gay and then just plain gay. He'd previously mentioned having a wife, but in a flashback their relationship was played out as a reference to Cat on a Hot Tin Roof, so...
Milhouse: A lot of jokes travel this route; Bart's Heroic BSOD after disastrous early school experiences (flashback episode "Lisa's Sax") lead to this exchange with a school counselor:
Dr. Pryor: Mr. and Mrs. Simpson, there's nothing to be alarmed about. Public school can be intimidating to a young child, particularly one with as many flamboyantly homosexual tendencies as your son. Marge: Bart's gay? Dr. Pryor: Ah, whoo, wrong file. (Counselor replaces file into its rack; audience sees the label: "van Houten, Milhouse").
Skunk Fu!: Baboon is known to behave very effeminately at times, the hand gestures, the hip swings, the fact that he was willing to teach a monkey how to act like a girl and dressed up like one for Dragon's birthday, on the other hand he has been shown to have a crush on Fox the youngest shapely female character in the show.
The Smurfs: Vanity Smurf is very effeminate, has the stereotypically affected voice of a "gay man", has a flower on his hat, and doesn't care about anyone but himself.
Eric Cartman and his obsession withKyleBroflovski. The subplot of the three-part-special Imaginationland consisted of Cartman trying to force Kyle into sucking his balls, to the point where he traveled across the country to get him to do it, and the episode ended with Cartman actually imagining him doing it, complete with calling him a 'dirty girl'. Additionally, Cartman states that girls are vile and disgusting, had a relationship between Ben Affleck and his hand puppet of Jennifer Lopez, and he made-out with a cardboard cutout of Justin Timberlake, while he was pretending to be Britney Spears. He performed oral sex on Butters, and made Butters reciprocate in a feeble attempt to right the first blowjob, which just lead to Butters being sent to a camp for gays.
Stan and his funny (as his mother puts it) relationship with Kyle makes Cartman look straight in comparison, though whether his girlfriend makes him this or Ambiguously Bi depends on how serious he actually viewed that relationship (it was never more than shallow, and it took years before Stan stopped vomitting uncontrollably around her). Trey said over 10 years ago that of any of the four main boys, Stan is the most likely to turn out to be canon gay, because he's the most sensitive. You can still see it.
In "Two Guys Naked in a Hot Tub", Stan's parents try to get Stan to play with other kids while they're at a party. Randy says to Stan that he has to make more friends. "You can't just spend all your time with Kyle, people might think you're... funny."
Kyle himself might be of the Armoured Closet Gay variety; he's the only male of the main boys who expresses disgust at naked males, when the other male student characters seem fine with watching naked dudes, implying that he is unsecure about his own sexuality; furthermore, the only things he considers disgusting are suspiciously penis related (pee and bananas), while he doesn't have any problems with far less hygienic things like anthropomorphic sapient shits, further implying that he wants to make himself look straighter even though he has no need to.
Terrance & Phillip's situation is so ambiguous even they don't know what it is:
Terrance: That Scott really hates us. Phillip: Yes, perhaps he's homophobic. (Silence) Terrance: But we're not gay, Phillip. Phillip: We're not?
A running gag with Mr. Garrison for several seasons. He's virulently anti-gay, yet sports a stereotypical gay lisp. Several characters state outright that they thought he was gay, but he insists that he just acts that way to "get chicks". In one episode he writes erotic fiction and focuses on penises, seeming to get very aroused by his own writing, but then insists that he's just writing to what his audience, women, want to read. In later seasons he comes out of the closet.
Jimbo and Ned. They're unrelated, live together, evidently have hospital visitation rights worked out. And then, Jimbo can say "fag" without getting bleeped, which according to Mr(s). Garrison means that he is one. All the other guys at the bar are bleeped when they say fag. After an awkward silence, Mr(s). Garrison says "Well, we've certainly learned something about Jimbo today."
Butters. He has been shown crossdressing, he's feminine, innocent, submissive, polite, and fits The Twink status. While he has shown affection toward girls, it's mostly blocked by his naivety. Oh, and he's more than willing to share his "creamy goo" with all the boys.
Kyle and Salty Mike from Squirrel Boy are mostly seen together. They also live in the SAME HOUSE!.
Storm Hawks: Arygyn the Skeelur, in Episode 25, "The Lesson", Arygyn (voiced by Scott McNeil) appears to the Storm Hawks team to train them. He is rather flamboyant in his mannerisms and clothing style, and often makes comments about someone's lousy hairstyle or the need for more decorations on the Condor.
Symbionic Titan: Todd the decorator of the student council from the episode "Under The Three Moons". He wears a pink shirt, tight pants, and a scarf, he's very flamboyant, speaks with a lisp, and skips when excited.
Time Squad has Larry 3000, especially in the second season. The first season did have its share of gay innuendo with Larry, but not as much as Season 2; it's like the writers knew the show was getting cancelled and they wanted to get as much as they could past the censors. The biggest example being "Ex Marks The Spot", an episode that was pure Ho Yay with Tuddrussel and even ended with the line that he would be "sleeping on the couch tonight"; the nightclub scene from "Day of the Larrys" is a close second.
Blitzwing, or more precisely, one third of him. When he's in his "Random" persona he has a high-pitched giggly voice, enjoys flamboyant dancing, is "light on his oscillating servos" and on one occasion referred to a crane as "big, bold, and sassy" — before he realized that the crane was an inanimate object and not a fellow Transformer. Blitzwing's other two personas have not expressed any sexual preference, but he seems actively repulsed by Blackarachnia. However this may be more because of her technoorganic form rather than her female persona. Oddly, while his other two personas seem actively repulsed by Blackarachnia it is the Random persona who wanted to "express his feelings for her in song". Said song being The Itsy-Bitsy Spider.
Starscream, who, as well as being on occasion quite flamboyantly fabulous, made five clones meant to represent different aspects of his personality — one of which was distinctly female. Which part of his personality was she? The answer given was "guess".
Just sounding like a whiny little girl doesn't make this trope, but G1 Starscream was a tad that way too - the most infamous moment here is him glomping Megatron.
Generation 1 Tracks is famously this. Fan constantly either joke or say that he's secretly gay despite the fact that Word of God says this isn't true (or at least wasn't intended).
Tutenstein: Luxor the cat shows signs of this in the "Cleo's Catastrophe" episode where he thinks about dating Jake.
Ugly Americans: Twayne is deliberately written as an ambiguous homosexual (or at least bisexual, given his attraction to Callie) as a running joke. He embodies a number of stereotypes about gay men, such as watching Oprah, being a poor athlete, and is even shown wearing a dress as a child. Additionally, most, if not all of his close friends are women and he even belongs to an all-female social club called "The Girls Club".
Watership Down: Cowslip from the animated adaptation acts like an elderly gay gentleman. He gets very excited when Hazel says they have no does (female rabbits) with them; he also hates getting wet and often brushes his ears back like an effeminate man would do with his hair. He was possibly intended to be more "intellectual snob" than gay, but that's what the viewers saw there.
Carver is into fashion, especially shoes but also mostly all of it, and gets caught wearing and/or buying women's clothes repeatedly, claiming they're for his sister.
Tino comes from a broken family, dominated by his trying-hard-to-be-unorthodox mother, and also has a very submissive/dependant personality that hints at a back-burner relationship or fascination with the local bully rough trade (as hinted at in said bully's not-declined offer of "Take me to the concert, and I'll beat you up" when Tino found that he had an extra ticket for a local concert... Tino's response was "I'll think it over").