Grif of Red vs. Blue spends the last moments of the third season screaming "No-!" (seemingly for hours, as said by Donut.), after the Red Team returns to Blood Gulch after an endevour with O'Mally. This extends to the the entire first episode of the fourth season, with lots of ridiculous plans (devised by Sarge, who else?) that attempt to force him off the cliff he's perched on. The plans are, in order, allowing him to scream until he passes out.
Grif: NOOOOO! Sarge: Simmons! Find a way to get Grif back down here right now! Simmons: Alright. HEY GRIF! SHUT THE FUCK UP AND GET DOWN HERE! WE NEED TO CHECK OUT THE BASE! Grif: NOOO! NOOOOOO! NOOOOOOOOOOO! Simmons: I don't think he's listening, sir. Grif: NOOO! THAT TIME I WAS ANSWERING YOUR QUESTION! NOOOOO! Sarge: That does it, Simmons, go get him down now. Donut: Sarge, if he keeps screaming like that, he'll pass out, and fall off the cliff! Sarge: Cancel that order Simmons! Grif: NOOOOOOOO! Sarge:(sighing) Donut, get me a sniper rifle.
Gets played much more seriously later in the series: in Season 8's penultimate episode, from Church when The Meta stabs Tex and extracts her into an AI memory unit; in Season 10's episode 16, from Alpha-Church, when Sigma (as The Director) tells him Tex is dead; and episode 19 of Season 10, from Carolina as the newly-"born" Meta rips the AIs Eta & Iota out of her skull, and from Tex when The Meta proceeds to throw Carolina over a cliff to her (presumed) death.
Strong Mad gets one of these in an encounter with Senor Cardgage on Homestar Runner.
Marzipan gets to say one after being forced to become Coach Z's girlfriend in "Doomy Tales of the Macabre". Since she read the cue card incorrectly, her first attempt was pronounced with a long "oo" and sounds more like "moo" with an "n" than "No!"
In the Eddsworld episode "Zanta Claws II", Tom has only recently gotten a present of reality eyes. Zanta however is about to kill the real Santa. Tom takes the shot while letting off one of these. The shot hits his eyes, reflects off them and hits a chandelier, crushing Zanta at the cost of his eyes.
Tom: I hate Christmas...
In 'The Camping Webisode' of DSBT InsaniT, Koden does this when he discovered the groups campsite has been ruined, and Duck when Martha accidentally pops Balloon.
In "Gelatin Swingsets", the Cutie Mark Crusaders are dismayed to discover their coupon for a free gelatin swingset has been taken away, and Scootaloo lets loose a Big No. But since she only speaksscat, it comes out as:
In the opening of "The Penny and Clyde Show", the big no pulls double duty as an expression of dismay and and answer.
Pinkie: Nooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo... Applejack: Dammit, Pinkie, would you just shut up? Pinkie: Noooooooooooooooooooooooooooooooooooooooooooooo—
Sage from "Shadows Of The Past" in episode 20 does this after Eieon mentions they should get Doc and Pinky to help them study the giant computer they found in the humming box. Justified after the ordeal they just went through with two assassins and a giant killer mech one year old freak child. It Makes Sense in Context
Parodied in episode 8. Snowball does this again when he isn't chosen for the relay race. This time, it is sped up to save time.
Justified in episode 17, when Leafy accidentally shatters Ice Cube with a frisbee throw and recovers her after answering a long string of questions regarding the recovery, only to shatter her again just before the sun goes down. This causes them to fail the challenge.
As well as when someone steps forward for his part in a barberhsop quartet and hears a click, followed by 'Hello!'.
"I'm going to punch your face... in the face." "NOOOO—(zoom to face within face)—OOOOO!"
Played for Laughs in the first episode of RWBY's second season during a massive food fight between RWBY and JNPR. Weiss has just been knocked out during her duel with Nora, who smashed her right into a pillar at one end of the cafeteria with her melon hammer. Ruby cradles her in her arms in the style of Pietà Plagiarism and screams at the top of her lungs as the pillar she was smashed into collapses around them, acting as if Weiss had just been killed in action. (Of course, this doesn't actually mean that Weiss is going to die, as she's shown to be just fine seconds after the food fight.)