In fiction, a Twitchy Eye is never merely a facial tic — for some reason, a twitch reveals your state of mind.
The prevailing notion seems to be that an eye twitch denotes insanity, which can apply to a permanently mad person with a permanent tic, or an unstable character whose twitch manifests along with their madness. As such, it usually accompanies a Broken Smile, and sometimes overlaps with Mad Eye.
Of course, there are other emotions that give characters a Twitchy Eye, including anything from extreme annoyance, to horror, to caffeine. so much that *twitch twitch* has become a standard sound effect in anime and manga, as well as a standard anime gesture of slowly raising a clenched fist with a twitching eye. Might be the prelude to a Villainous Breakdown or a Heroic BSOD.
Other times it's not a mood, but an action — such as lying — that will bring about the twitch.
In Real Life, a twitching eyelid can be sign of emotional instability, but it's usually a sign of tiredness or severe stress.
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Anime and Manga
Yu-Gi-Oh!: Bishounen Duke Devlin gets a twitchy eye when he's losing to Yami at a game of Dungeon Dice Monsters (Which he invented). Ouch.
Kagami does this occasionally in Lucky Star, when she's trying to deal with Konata.
Kaname gets this a lot in the high school parts of Full Metal Panic!!. Mostly due to Sousuke.
The otherwise unflappable Xellos got an eye-twitch in Slayers TRY after Filia called him "raw garbage".
Natasha Zabikov gets one on G Gundam when a boy calls her "Old Lady."
Sir Ruber of the movie Quest for Camelot, one of the characters with a permanent tic, starts out with a slight Twitchy Eye that gets worse and worse throughout the film, until his whole face is practically quivering.
Chief Inspector Charles La Rousse Dreyfus in the Pink Panther movies.
Ed the hyena from The Lion King. He's the least sane from their pack and quite a Cloud Cuckoo Lander.
The anchorwoman in the film version of V for Vendetta was said to blink involuntarily when she reported a story she knew was a lie.
Several characters played by Kitano Takeshi have a twitchy eye, most memorably Nishi in Hana Bi, whose eye twitch announces an imminent outburst of sudden violence.
The Incredibles: after Kari has been putting up with Jack-Jack throughout the whole of the movie, she manages to freak out the new 'babysitter' with one of these.
Russell being dragged across the Big Bad's window induces a slight eye twitch in the latter in Up.
In The Kid, while trying to make a sandwich for his younger self, Bruce Willis's left eye starts to noticeably twitch.
Amélie's mother has developed an eye twitch as a sign of shot nerves.
Seed Of Chucky: Glen, who is the genderless offspring between the eponymous killer doll Chucky and his dollified girlfriend Tiffany, always has a notable twitch in his/her eye whenever he's anxious or goes into an Ax-Crazy episode. He also tends to wet his pants.
In the novel The Golden Goblet, the main character's villainous half-brother has a permanent tic in one eye that makes him look even more sinister.
Mr Teatime in Hogfather has an odd twitch whenever his name is mispronounced.
In Jingo Lord Vetinari tells Vimes that the reason he doesn't like Captain Carrot in the room when Vimes is being debriefed is that Carrot tends to eye-twitch whenever Vimes tells a direct lie.
-Vetinari: I just can't stand to see his poor face twitch all the time.
Prince Andrei Bolkonsky developed facial tics in War and Peace, but that was probably due to a degenerative disease. The way it's written sounds like his personality (he was a curmudgeony sort) was getting the best of him.
The First Law: Glokta's ruined eye (along with his face, sometimes) tends to begin twitching/blinking uncontrollably when he's nervous, lying, or any other time it's inconvenient to look like he's not utterly composed. He Lampshades it in his thoughts, calling it "treacherous jelly."
In the novelization of Resident Evil 0, it's mentioned that Birkin's paranoia has caused him to develop a facial tic. Though, it's a slight subversion because it's actually on the corner of his mouth rather than his eye.
Barney in How I Met Your Mother does this occasionally, most notably when he was trying (and failing) to hide his fear at being on the receiving end of another slap from Marshall on Slapsgiving.
Peter on The Monkees sometimes displays this tic when he is nervous or startled.
Davy: "Are you nervous?"
Peter: "Nervous? Don't be silly! Look at how slowly I'm twitching."
On Just Shoot Me!, Dennis realizes Nina is trying to keep a secret because of her "I've got an itch to snitch" twitch.
Buffy the Vampire Slayer: Xander briefly developed an eye twitch every time anyone mentioned Faith, after they'd had sex together.
Boy Meets World: Topanga begins to develop a really nasty one during the preparations for her wedding to Cory, which, naturally, are full of wacky hijinks as Eric... "helps."
The Big Bang Theory: Sheldon has this (among other tics) when things don't go his way (for instance people interrupting him when he wants to show off his knowledge, or not being able to answer a question to a quizz).
On The Nanny, C.C. Babcock has it happen whenever she sees Fran Fine and Maxwell Sheffield getting along. It first occurs in season one's "Here Comes the Brood".
Fran: (to the Sheffield children) Now everybody say goodbye to Daddy...
C.C.: "Daddy"? Did I miss the wedding? (her eye twitches)
Count Veger in Jak and Daxter gets one when he finds out the Precursors are ottsels.
A twitchy eye is one of the first signs you get that there's something that's not right about Lisa in Silent Hill 1.
Portal 2 gives us a robotic example in Wheatley the Personality Core. He gets damaged early in the game and, from that point until the showdown with the Disc One Final Boss, he has a short circuit that randomly causes him to spasm and give off sparks. Since his body is intended to resemble a robotic eyeball inside a frame, the effect is strongly evocative of a twitchy eye in a human. This actually works as Foreshadowing that Wheatley is not as sane as he appears to be.
In Mario Strikers Charged, this may happen to Luigi if a ball (or more than one ball) gets in the net. Yes, Luigi.
The OCD-suffering Hannelore of Questionable Content does this when Marten unthinkingly jokes about spreading garbage around her apartment.
This is supposedly the "tell" in poker of the Patterner in Exterminatus Now. To clarify: the Patterner is an Expy of Tzeentch, Warhammer 40,000's god of Magnificent, manipulativebastardry. When he's shown preparing to deal, he has at least five decks of cards floating around him and held in all four hands. And he displays he's bluffing when his eye (which is not only all of his head, but floats around separate from his body) twitches.
Drive: Nosh does this when he learns that he has to go to Tesskil, whose inhabitants, the Tesskans, lost a war with his own species, the Veetans.
Frankie And Stein's male half of the titular duo has an eyetwitch fairly regularly, generally when he has a wonderful idea. It also appears to be genetic, as it has been seen in his cousin. It may be contagious, as it has also been spotted in Karl's friend Adam, and the female half of the titular duo, Frankie.
Karkat gets one in the Alterniabound flash game, while demanding that Gamzee stop dancing.
The main character of Doctor Horrible's Sing-Along Blog has this as a Character Tic. It becomes very pronounced when he's nervous, almost to the point of being a Tourettes Syndrome tic. In particular, it tends to occur when he's discussing his Arch-Enemy Captain Hammer and when his desire for his Love Interest, Penny conflicts with his desire to be a supervillain. In contrast with the typical use of the trope, the tic becomes noticeably less pronounced the stronger he grows in his Dr. Horrible persona.
Any character in Invader Zim will get twitchy whenever they start feeling crazy, but particularly Gaz and the so-happy-it's-freaky experiment, Nick. For many of the other "normal" humans, it can be interpreted as showing how the stress of living in such a Crapsack World has them perpetually near the breaking point, such that they twitch whenever something pushes them just a little bit further.
When he appears in Poker Night 2, this is one of his "tells" to indicate he has a bad hand.
Tweek of South Park, in addition to his overall jitteriness, has a perpetually twitchy eye.
In a ''Simpsons'' parody of Amadeus, Lisa, as Salieri, does this when the emperor declares all music other than Beethoven obsolete, making her schemes against Mozart all for nothing. She then hails a coach for the nearest asylum and rides off, cackling hysterically.
If you pay careful attention, you can see Ned Flanders twitch an eye during "Hurricane Ned" after the rebuilt house falls down, just before he puts his glasses back on and completely loses his shit.
Yet another Simpsons example involves Moe the Bartender. When he attempts to remake his bar into a family restaurant and himself into a more family-friendly image, the customers—as per usual for Springfield—quickly annoy him to the breaking point. What sets him off on a G-RatedCluster F-Bomb barrage is a child complaining, during Moe's twitch of anger, that her soda was too cold.
An episode of Futurama ends with a shot of Fry doing this after being forced to destroy his robot Lucy-Liu girlfriend, and then watch Bender start making out with the real Ms. Liu. Or at least her head. It's complicated.
Megatron of Transformers Animated no doubt appreciates Lugnut's unswerving loyalty, especially in comparison with Starscream's constant backstabbing, but once in a while gets a twitching optical sensor when Lugnut overdoes the fawning adulation. "Just...go." * twitch twitch*
Earlier in the franchise, Galvatron starts to exhibit a serious twitch towards the end of the fight with Hot Rod in Transformers: The Movie. Justified, given that his day has gone quite south, he's had his psychic leash yanked for much of the movie, and he later has a reputation for being, well, just plain nuts.
Megas XLR has Magnanimous get one after being sucked through a portable black hole. And he lampshades it.
Guess how Kevin can tell that his friend Ben is lying? It even becomes a minor plot point in one episode.
Happens often in The Fairly OddParents; one example was when Timmy's mom showed him that her old bikini still fit.
On Jimmy Two-Shoes, Heloise does this after Cerbee and Beezy ruin her private picnic with Jimmy. She then is paralyzed with rage, breathing deeply and heavily in one spot for hours.
Atomic Betty: Maximus IQ does this after Galactic Guardian HQ is destroyed, because Noah and Minimus accidentally do it before he can.
The lunch lady in the Fanboy and Chum Chum episode Little Glop of Horrors does this when Fanboy insults her glop.
Not to mention Lenny, who even has an anti-stress twitch cream.
Miss Achmetha from VeggieTales has this as a character trait, and comes up pretty much every time she's used.
A subtle one in Duke and the Great Pie War done by Duke (played by Larry):
In "Suited for Success", Rarity earns one of these when she learns the famous fashion designer Hoity Toity is going to be attending her fashion show, and seeing the (tacky and overdone) dresses she made for her friends. She does it again at the end, when Hoity Toity agrees to sell her fashions at his shop, and proceeds to place a big order.
Twilight briefly does this while worrying about whether Princess Celestia will approve of her friends in "A Bird In The Hoof".
The Tick has the character of Big Shot, a No Celebrities Were Harmed version of The Punisher. He gets all eye-twitchy whenever he shoots, fights, or even says the word "gun". This is indicative of some pretty deep-seated mental trauma, as he is prone to collapsing into uncontrollable sobbing while screaming "Why didn't you love me, Mommy?!"
Spongebob Squarepants does this when Patrick meets him in his house. "I'VE BEEN WAITING FOR YOU PATRICK!"
The Powerpuff Girls: Buttercup does this in "Nuthin' Special" in a scene where her sisters keep matching her in showing off special powers.
Roodaka's eye twitches in BIONICLE 3: Web of Shadows as she expresses her demand for the corpses of the Toa.
"Opsoclonus" is a term that just broadly refers to any kind of uncontrolled eye movement. A lateral twitch is specifically called "Nystagmus" and can be a sign of an underlying neurological problem if it persists.