Necro Critic did this when he found out there was more to Ehrgeiz than just the terrible fighting game. This one lasted past his 30-second long end screen and continued even after stopping for air.
The end of the season 3 finale of AH.com: The Series: after it's revealed that they inadvertently overwrote the location of the Planet of the Nymphomaniacs in the ship's memory banks, the entire crew lets out a Big No so loud it literally echoes off distant galaxies and is heard in other dimensions.
A popular internet meme is RAEG, where a template comic strip is drawn on, with the ending of a grotesque face shouting "FFFFUUUU—" in situations like a no smoking sign, being caught watching porn, etc.
The Leet World's Chet usually greets the death of his drinking buddy Player with a cry of "Player, NOOOOO!" However, since the show is set inside a Video Game, dead characters are usually back in action next round. A notable exception is when Player is injured by Ahmad's grenade, as this took place outside the challenge levels where death is permanent. Lampshaded in the episode "Thunderhead" when Player breaks cover runs into the line of fire. Westheimer shouts "Player, No!" and Chet comments that it sounds better when he says it.
In the Zero Punctuation review of Spore, Yahtzee delivers a deadpan Big No. Twice. Both times in his inimitably snarky manner.
"So can his new game Spore possibly live up to that legacy? In short, no. In long, noooooooooooooo..."(total length: 17 seconds)"...ooo. So I guess what I'm asking is, 'Can brightly coloured monstrosities really compare to Batman?' Short answer, Nooooooooo..." (interrupted by closing music)
The Nostalgia Critic has "I HAVE A NEGATIVE RESPONSE TO THIS SCENARIO!!! I HAVE A NEGATIVE RESPONSE TO THIS SCENARIO!!!" This while making fun of Tombstone's Big No. However he actually lists this trope as one of his favorite clichés.
Rob (dressed as Emperor Palpatine) tells Doug (dressed as Darth Vader) that he must review the 1999 animated movie The Legend of the Titanic. Guess the result.
At the end of The Odd Life of Timothy Green, when the Critic hits his Rage Breaking Point...again:
Critic: NO! [pulls down technicolour swirly image over the camera, bearing the word "NO!" in big red letters, then waves a hand in front of it] NOOOOOOOOO!
Nostalgia Critic also discussed it in the Top 11 Coolest Cliches as "Screaming 'No!'"
In The Batterys Down Courtney has one of these when Jake says he's quitting musical theatre.
Also from a Let's Player, Chuggaaconroy's occasional Epic No. To be fair, it isn't a long, drawn out "NOOOOOOOO" as much as a deep, harsh "NO!", which in context is a rejection, not the cry of despair seen in movies/other media. Chuggaaconroy parodies himself in this video at about 11:46. "And there's Luigi's Epic NOSE!"
Though he does have "cries of despair" frequently, particularly when he's joined with the rest of The Runaway Guys.
Two in Water Human, both very dramatic and ending abruptly. First, the Water-Human cries out over David Hasselhoff's corpse, before realizing that it's weird to angst so much over a guy he knew for less than a minute. Later on Water-Human and a prison warden cry over the Large Beetle, who claims to have a heart attack, but then reveals he was just kidding.
Marik: I'll just work on my tan. Wait. I'm a vampire. I no longer have a tan. NOOOOOOOO!
The end of the "Sloppy Roethlisberger" episode of Epic Mealtime has Harley accidentally breaking and spilling an entire bottle of Jack Daniels, with this being the appropriate response from someone in the background.
A couple of times throughout Nakar's Let's Play Ultima series, but most memorably right at the end of Ultima VI.
Beh Lem: "To be saying nooooooooooooo in slow motion!"
Tom Ska has a NO! moment while discussing the post-production of the then-impending video "Holy Shit, Cats!"
Tom: ...then I have to send it over to Andrew, who puts the special effects in—... Jenny: *produces Tom's "Dubstep Sunglasses" and puts them on. Pop music plays instead* Tom: NO!! NOOO!! NOOOOO!! *video cuts to the end*