In Anchors Aweigh, two Navy sailors take a runaway kid back to his home, where he lives with his aunt. They think she must be old, and are surprised that she is about their age.
It's never explicitly stated what Niles expected in Frasier, but he is clearly surprised the first time he meets Daphne, mentioning that Frasier told him she was English and um, well... He most likely assumed she was a stuffy older lady.
In Tower of God this happens commonly since the Shinsoo atmosphere slows down and sommetimes even stops aging. This leads to cases of Ship Leesoo being in his late twenties and Yeon and Hatsu in their late teens whereas Androssi is about 300 years old and her "niece" Anak is even older.
In Alvin and the Chipmunks, Alvin hears about having an oldest fan, and thinks she's just in her twenties, while she is actually a senior citizen.
In the Baby Sitters Club movie, the parent of one of Stacey's baby-sitting charges warns her in advance that her "little cousin" from abroad will be home when she comes by to baby-sit. She brings with her a little American flag to give him, expecting a young child. Instead, he's an extremely attractive 18 year old.
In the Fantastic Four, the Thing frequently mentioned his 'Aunt Petunia', with the implication that she was an old woman. When she finally appeared in the comic during John Byrne's run, Petunia (she prefers 'Penny') was revealed to be his uncle's second wife and an attractive woman about the Thing's own age.
In Mary Renault's The Last of the Wine, the main character's lover has a spazz attack when he realizes that the woman the main character has been calling "Mom" for the whole book is a stepmother and about the older lover's age.
In an episode of ER an injured toddler comes in and shouts that Uncle Charlie hurt him. Someone asks if they should call Social Services only for someone else to point at another, unharmed but still grouchy, toddler, and say "That's Uncle Charlie".
In Denno Coil Haraken's aunt, who is referred to offhand as being the commander of the Satchiis, is assumed by the main characters to be at the very least middle-aged. When she finally appears, it turns out that she's actually on the nearer side of twenty.
In Indiana Jones and the Temple of Doom, Willie Scott dresses up beautifully for the dinner at Pankot Palace in hopes to impress the wealthy Maharajah. She's chagrined when he's revealed to be a kid around the same age as Short Round.
In an episode of United States Of Tara, Kate's boyfriend is about to meet Kate's family, and Kate had previously mentioned that she had a younger brother. Kate's boyfriend buys a toy train, expecting that he'll be quite young, except Marshall (Kate's brother) is in his teens, causing an awkward interaction.
It's a running gag in the Baccano! light novels that, despite nearly half the cast being immortal, only Firo (already a bit young-looking before immortality) has to deal with the annoyances of not looking his age, such as getting carded at age ninety, being detained for claiming he was seventy on his passport, and constantly being called 'babyface.'
The protagonist of the first story in Himitsu Kichi mistakes a sixth grader for a college aged student. In turn he mistakes her for a kid his age when she's 16 years old.
In an episode of Firefly, Mal invokes this trope to evade I Never Said It Was Poison, when asked whether he has seen fugitives, "a brother and sister", by pretending to assume that the pair in question were children, when he actually knows exactly who and where they are.
Early in Aunt Dimity: Detective, Lilian Bunting asks Lori to entertain her nephew "Nicky" while she and her husband attend Prunella Hooper's inquest. Lori expects to receive a visit from a small boy, and is surprised when thirty-something Nicholas Fox introduces himself.
In Lucky Star, Miyuki's mother looks so youthful and cute that she's actually often mistaken for her older sister rather than her mother.
One Oil of Olay commercial had a stage psychic guess a woman's age, and undershoot because she was using the product. She plays along, and says the psychic was correct.