Pinkie's entire tutorial, fourth wall breaking and all. Twilight's reactions make it even funnier.
Twilight interrogating Applebloom:
Applebloom: "I had homework to complete for school after that, so I was pretty much doing that for the rest of the day." Twilight: "That took up the WHOLE DAY?" Applebloom: "Twi, you have NO IDEA how scary Miss Cheerilee can be in the classroom." Twilight: "Do tell." Applebloom: "After Family Appreciation Day, everypony in the whole class had to write an essay on how it's bad to fall asleep in class when somepony's talking." Twilight: "That...doesn't seem so bad." Applebloom: "Oh, it ain't, 'cept for the fact she wanted us to explain our position on why it's bad and what 'bad' is using classical philosophers' arguments as a basis. Jeremy Benthoof will haunt me to the grave, Twi. TO. THE. GRAVE. Twilight: (This is an ELEMENTARY SCHOOL?)
While interrogating Rarity about what she saw the night True Blue and Opal went missing, Twilight can suggest that the one who stole True Blue was Opalescence. Rarity promptly asks her to retain a grip on reality.
Really, any of the obviously wrong or obviously stupid answers can be funny.
The first time Twilight pulls off an "Objection!" - well, the pose; she doesn't actually say it - Apple Bloom asks very incredulously what she's doing. After a brief back-and-forth, Twilight internally groans and basically says "Okay, I get it, I'm not a lawyer!" and then proceeds with the actual investigation.
Talking with the guard to the Diamond Dog caverns. When asked to enter, he simply says "No." And the background music just stops.
After that, he always responds with a one-word answer (almost always "No." again, though if the situation calls for it, he occasionally says "Yes."), and every time, the music stops again.
Twilight being so utterly confused that the Diamond Dogs are so intelligent in legal matters - they demand a search warrant, they refuse to talk about True Blue because the search warrant is for finding Opal, they know about "innocent until proven guilty", and more.
Twilight: Did they steal books from my library too?!
Twilight (internally): I can't tell if they're smarter than they seem, or dumber.
What makes the above conversation is the way Rover says his last line. Every other time you talk with Rover, he's either yelling at you, being a Smug Snake, or getting briefly scared by Rarity or by you succeeding in your final confrontation with him. But in that one line, he says it like a kid saying ice cream tastes good. Even better since in his previous line, he was practically ranting at Twilight.
Asking any of the Diamond Dogs about Rarity produces great stuff.
Sweetie Belle gives a quite impressive description of the deep meaning behind the CMC cape design. And why the gold interior? "It was pretty."
Scootaloo's impenetrable Squee if you ask her about Rainbow Dash.
Investigating the distinctly human-styled rope swing on the CMC clubhouse gets Twilight fascinated about the apparent lack of physics involved. And if Apple Bloom is with you she jumps right on, interrupting her thoughts.