Beatrice: Are you here to make fun of me too?

Kay: No ma'am. We at the FBI do not have a sense of humor that we are aware of.

Plastic Man: Was that a joke?

Batman: From a ruthless, obsessive Creature of the Night? Don't be absurd.

— Formerly Known as the Justice League

"This isn't funny, Dean! The voice says I'm almost out of minutes."

— Castiel (talking on a cell phone and standing on a street corner), Supernatural

Dean: Cas, I'm human, I need sleep. Just give me, like, four hours, man.

Castiel: ...Four hours? *Dean hangs up on him* I'll just... wait here, then. (Proceeds to stand perfectly still and stare off into space)

—Supernatural

Garak: Mister Worf, you're no fun at all.

Worf:...Good.

— Star Trek: Deep Space Nine

"I am not interested in having fun."

— Tuvok, Star Trek: Voyager

"EDI has a great sense of humor. Or at least a really funny lack of one"

— Kasumi Goto, Mass Effect 2

Jade: (to Billie) Okay, okay, sometimes you’re no fun at all.
Ayla: * walks in* And speaking of no fun at all, I’m here too.
Billie: But I’m more fun than Ayla, right?
Jade: Oh yeah, everyone’s more fun than Ayla.
Ayla: In fact, I’m changing my name to… No Fun Guy!
Billie: Hey Jade, that could be like your arch-enemy.
Ayla: That’s right… Giggle Girl! Once I unleash my latest invention, the Federal Tax Code Emulsifier, no one will be having fun ever again!
Jade: Giggle Girl doesn’t know the meaning of the word defeat!
Ayla: And she doesn’t know the meaning of the word ‘rationality’ either!
Billie: Or the meaning of the word ‘crayon’!
Jade: Giggle Girl does so know the meaning of the word crayon! It’s the cute little pink thing I use to write ‘No Fun Guy is a big poopyhead’ all over your secret lair.
Ayla: Doh! I knew I shouldn’t have shot all those henchmen even after they said they didn’t do it…
Jade: Hah! Giggle Girl always defeats the forces of boredom and no-fun-ness!
Ayla: But can she defeat… your next final exam!!!
Jade: Noooo! Not that!
Billie: (announcer voice) Has No Fun Guy defeated our heroine? Can she find a way out of this hideous trap? Tune in next week for the further adventures of GIGGLE GIRL!

—Whateley Universe

Lord Downey: "Is this a joke?"

Auditor of Reality: We have no appreciable sense of humor.

—Hogfather

Krusty the Klown: Free comedy tip Slick, the pie gag's only funny when the sap's got dignity.
[Sees Sideshow Bob]
Krusty the Klown: Like that guy! Hey Hal, pie-job for Lord Autumnbottom there!

I will not suffer the same fate as cream filling!

"I am the only serious character in this show. That's the joke."

Jack: Hey, Optimus, wanna see something funny?

Optimus: No.

Angel: I'm brooding.

Lorne: You're watching hockey!

Angel: Yeah, but my team is losing.

Angel: I do not have puppet cancer!

It's hilarious how you have no sense of humor!