Wolf Link stands fiercely at the entrance of the deserted town at high noon. Sweeping camera angles and quick cuts map out the area and display the many cats that now infest it. Then the spaghetti western music cues, and you know that Link is going to befriend the hell out of them.
Midna is a veritable vending machine full of these. It helps that she's the game's resident Deadpan Snarker, too. One quote in particular jumps to mind:
Midna: Hmm... I don't suppose there's any nicer way to hunt these things (light bugs), huh? Well, you had to sacrifice someone's house to find Tears of Light...but that's how the cookie crumbles, right?
Another one, in a nice case of either Lampshading or some Fourth Wall Trope:
Midna:You're just now figuring out where Zant got his power?
There's also her antics with the sword and shield once you go into the Twilight for the second time. She tries to hold the sword by the blade, and wears the shield like a mask. This was even referenced as an alternative costume in Hyrule Warriors!
"Say something. Am I so beautiful that you've no words left?"
If you find the bridge that goes to Kakariko Gorge, the missing piece of the Eldin Bridge, or the ancient cannon, but teleport to the wrong place, you may get this gem (among others possible):
Midna: Hey! Are you doing that on purpose to make me mad?! Are you trying to drive me crazy, bringing me to a place like this?! Give me a break!
In another case of in-game lampshading, when you enter the Lakebed Temple, she says that it's a much better place than the Goron Mines. As you make your way through the temple, her opinion changes from enthusiastic to annoyed.
Midna: Why did those Zoras think it was a good idea to build such a huge temple?!
Whenever Link turns into a wolf, she gets on his back quite abruptly...every single time. The annoyed growl and look he gives her are quite amusing.
In the manga, the first time she tries to bully Link around, he threatens to bite her. She just grabs him with her hair-hand and zaps him.
Right before she transforms after using the Fused Shadows, she is flung against the wall a few times by their power before flying offscreen.
A funny part is when Midna calls Zelda "Twilight Princess". Ironic, because Midna is the real Twilight Princess.
In fact, even crazier is that if you listen closely to Midna's random gibberish voice, it can produce many gems like these.
Similar to in Link's Awakening:
Trill: Pay like you're supposed to next time, cheapskate.
Try running around Castle Town in wolf form. It's funny when all the citizens think you're an enemy monster and they scream and cower before you, but it gets hilarious once all the town's guards start following you around, aiming their spears at you. Too bad they're too afraid to attack, especially since they, too, cower as you pretend to attack them.
While you're at it, do the spin attack in wolf form once all the guards surround you. They run away as fast as they come.
For more fun, enter the STAR tent as a wolf. "Hey! You idiot! Don't come over here! AH-CHOO! I finally get my canine allergy under control, and now...Ha...Ha...AH-CHOOOOOO!!"
Just when you think you've finished off Armogohma, the triumphant victory music suddenly stops, and the boss's less-than-impressive true form tries to escape to the sound of a xylophone...just so you can finish it off for real with whatever humiliating tactic you can think of — one of which is standing there dumbfounded for a moment, watching it try and get away, then letting a Bomb Arrow or two loose.
Even better; Link's expression during the reveal is pretty much a Flat "What." without the sound. Priceless.
Even even better, Link does his fancy slash-slash, twirl-twirl-twirl-sheathe before realizing that this thing isn't dead... except that Armogohma is only vulnerable to the Dominion Rod-controlled statues, which means that Link drew his sword for the express purpose of twirling it all fancy. And he hasn't even let go of the handle when he realizes Armogohma isn't dead!
Try the Dominion Rod; it even grants an extra cutscene showing Armogohma being literally flattened by the statue's hammer. It looks like it got run over by the world's largest steamroller.
Castle Town Malo Mart.
Even better, the text you get when you buy the Magic Armor from the Malo Mart. "You got the Magic Armor! You bought it! You are smart! You bought it at Malo Mart!".
Even the dance they do is hilarious!
The Gorons at Malo's Kakariko Village mart are doing it!
If you look closely, you might notice half the people in Castle Town carrying Malo Mart bags.
One scene during the credits shows the entire town doing the dance in the square.
The very idea that Malo went from being a small kid who was jealous of Link's slingshot to the CEO of a store that bought out the most expensive shop in Castle Town (while still being a small kid). He's basically the opposite of Tingle in that respect.
Using your (strictly prohibited!) lantern in Barnes' shop. He wasn't kidding with those signs!
Barnes' reaction of throwing his hands up in disgust when Renado doesn't introduce him.
Barnes' interactions with King Bulblin; fearfully lowering his mask when KB raises his.
The intro to the third encounter with King Bulblin: You walk up to a boar in a dark room, you see two red eyes menacingly stare at you from out of the darkness as Bulblin looms forward with a massive axe...And then he casually backhands Link and the boar out the way.
In the desert area outside of Arbiter's Grounds, charging while riding one of those boar-like mounts causes Link to hold on for dear life, with his legs flying behind him.
When you have to revisit the Sacred Grove, Rusl calls over a golden cucco to help you get there. Once you've arrived, morph into a wolf and talk to it. It says "Twinkle twinkle little cucco, I am gold and not for you-oo."
Early in the game, you can hit Malo (the smallest of the three kids) with your slingshot and his head will just be knocked back.
One of the first things you can do in the game is go fishing with the rod that Colin made for you. There is a cat looking at fish in the river in Ordon Village. Catch a fish near said cat to gain its attention. Catch a second one and it'll run off with your fish. Link's expression says more than any words ever could.
Fight Ganondorf. Use your fishing rod. Now watch as Ganon stares at the rod, looking at the juicy worm... THEN STRIKE!
In Ordon Village, you can use some falconry skills to knock down a beehive... but for a funny scene, put it off until you talk to Hanch as he's throwing rocks at it. He manages to hit it... unfortunately for him.
Yeto's response if you turn down his offer to race you:
"You no like fun, uh?"
Also, Yeto excitedly (and violently) smacking Link to the ground whenever he gets a new ingredient for the soup. It happens so abruptly.
In most Zelda games that have Cuccos, attacking them too much makes them attack you. Not so here: attack a Cucoo enough (in either form), and the screen will fade to black. When it comes back, you are the chicken, albeit for a short while (This is not the case for the Golden Cucoo, or the Oocca.).
Zant's insanity before his boss fight leads to some amusing slapstick moments. His entire speech before the fight is hilarious as hell, if only because of his gestures.
In the Lakebed part of the battle, Zant appears out of the ground in a menacing giant version of his mask - then when you pull him out, he frantically tries to swim back.
When you knock Zant off the wooden pillars of the Forest Temple miniboss arena, he falls head-first into the ground, and gets his head stuck, cartoon-style.
During the Snowpeak part of your battle with Zant, he grows to a gigantic size and tries to crush you. To make him shrink and become vulnerable, you have to hit him in the foot with your Ball and Chain, leading to an amusing Agony of the Feet moment. And as he shrinks, his cries of pain grow ever higher-pitched.
Going back to Ordon village during the day lets you find the Postman hiding in the goat barn, feeding letters to the goats due to the bad handwriting in them. Similarly, he can be found within Zora's Domain desperately trying to find out who "Mr. Zora" is.
If you talk to Jaggle a second time near the beginning of the game after he's pointed out the hawk grass to you, he says:
"What's that stuff thinking, growing on a rock like that? I guess I just don't understand grass."
The entire battle with Ook is hilarious, not just because of his mannerisms, but because of one thing: his weak spot is his ass. You literally have to kick his ass to defeat him.
Alternatively, he's a monkey, and you have to repeatedly whack him in the behind to beat him. You're "spanking the monkey"!
Speaking of minibosses, what happens when you throw Dangoro in the lava is pretty funny.
One of the chickens in Ordon Village blatantly hits on Link in his wolf form. Yes, Link is literally a Chick Magnet.
Beth and Luda's little rivalry over who gets to take care of Colin after he saves Beth from King Bulblin. First they are taking turns wiping his forehead, doing it so often that Renado has to calm them down. Then, after you have returned Death Mountain back to normal and Colin is feeling a little bit better, they are trying to get him to eat and drink something. Luda with her stew and Beth with her specially mixed water (hot spring and sacred spring water combined!), constantly shoving it in his face. Poor fellow...
Any time Link talks to his Fangirls after he won the STAR minigame. The amount of Squee! is overwhelming.
EEEEEEK! It's HIM! ♥♥♥ KYAAAAAAAAAAA! *they run off*
Somewhat crosses with a moment of Awesome too. When you walk into the Goron's Elder Room, after having worked your way through the attacking Gorons onto Death Mountain, no fewer than four Gorons ball-up and start rolling towards Link, all at once. In the split-second before the Elder calls them off for ganging up on him, you can see Link posing himself to catch ALL of them. Guy's either well-earned that Triforce of Courage or he's overconfident.
After you defeat the Goron Elder in sumo-wrestling, talk to the Gorons. One speculates that they would've been sent flying! (Unlikely, but the mental image is amusing.) Another says something like "If I find out you cheated..." (Which you have, but nothing comes of it.)
After the Zoras recover, and you have the Zora Armor and Water Bombs (so you can get an item while you're there), sink into the pool in the Zora's Domain throne room. There's a Zora who's hilariously excited about the rock, swimming as if making up for the amount of time spent encased in ice.
If you blow up the rock you used to melt the ice within Zora's Domain, a Goron will come out of it. He was apparently taking a nap when lava encased him. While obviously bewildered on his sudden change in location (as well as the fact that he's in a "cool spring" as apposed to a hot one, which he admits that he likes), he gives you an extra bomb bag as a reward for freeing him.
Hena's reaction for you fishing a Piece of Heart while within her rowing boat.
Hena: What's that? A piece of heart?! Ooh, whose heart did you catch? You big sweetheart! Don't be shy! This one's yours to keep!
This sacred fish is only found near the Zora village. It... reeks.
The sheer fact that the Snowpeak Ruins, the dungeon of the Peak Province that resembles a mansion, happens to also be the house to friendly Yeti. You'd never expect that turn of events, especially since the Yeti are perfectly willing to help and give Link the mirror piece they had found, which was why Link followed one of them to their home in the first place.
Talking to Yeto as Wolf Link nets this gem:
Yeto: Uh! Rare color of wolf! Looks tasty... You want dinner, uh?
Yeta: No feel like meat tonight...
Yeto (disappointingly): Uh... That too bad...
If you talk to Yeto again,
Yeto: But... Looks so tasty, uh...
If you check the fruit stall opposite the Hot Spring Water stall in South Hyrule Castle Town, the item get jingle will play as Link obtains an apple... only to put it back, musing that he should look for the sweetest apple in the stall before buying.
Similarly, he can pick out a flower in another market stall, cueing the jingle... only to put it back, because no-one is manning the stall.
Also, in a similar vein, if you've reached the max number of rupees and get some from a treasure chest, it'll go through the jingle... and then Link puts it back.
After helping Telma and the gang get to Kakariko, in the cutscene afterwards, Link turns away from a job well done and gets a face full of Telma before quickly looking up at her face.
Later, Telma runs off with Renado, and afterwards you can find Renado trying his hardest to avoid her and her grousing about it.
The AI can be pretty funny.
In the City in the Sky, there's a room with two Lizalfos. Shoot one with a Bomb Arrow, and his buddy will run over to investigate. Or, more accurately, try to; he manages not to notice the gap in the floor, and falls right through.
More fun with Bomb Arrows and Lizalfos, this time in the Cave of Ordeals. There are four, this time. Kill one of the two closest to the center, then watch the others cluster where he was, allowing you to kill all of them with just one more, without even changing where you're aiming.
At some point in the game, you start seeing Zoras visiting the Death Mountain hot spring. It seems one is not particularly used to the heat...
Unlike previous games, the staircases in Twilight Princess have each step collision mapped. People who are used to rolling up the stairs may find themselves instead bumping their head and falling back, though going down is still perfectly safe.
The very first letter you receive in the game. From the postman. To flesh this out more, the letter basically says that if you see him running at you at lightning speeds, don't freak out and run away.
One of the rules of fishing: "Don't drown if you can't swim."
Yet another, "Don't cast your lures at other people." The very fact that people have actually done this before, which is the only reason why it would be in the rules, is downright hilarious.