Volunteer Matthew Harris, 24, spotted the copy of the first Beano Book, from 1939, as he sorted through items at the Cancer Research UK shop in St Andrews, Fife.
After the charity took advice from the book's original publisher, DC Thomson, the annual was put up for sale on behalf of Cancer Research UK by a comic book auction house.
It has sold for 4,264 – one of the highest prices ever paid for a copy of the book.
Tammy Kernohan, manager of the South Street shop where the book was found, expressed delight at the amount of money raised through the unusual find.
He said: "This is definitely the most valuable donation we have ever received.
"We're so grateful to the person who handed it in and to Matthew for discovering how much it was worth.
"All the money raised will go towards finding new ways to treat, diagnose and prevent cancer."
The book was published in 1939 and includes characters such as Pansy Potter the Strongman's Daughter, and Lord Snooty and Chums.
The popular character Dennis the Menace does not feature because he did not appear in the comic until 1951.
Malcolm Phillips, of Comic Books Auctions, said: "Our buyers rallied round to the most worthy of causes and I'm delighted that this exceptional example of the Beano annual number one has raised so much for the fight against cancer."
The book was bought by an anonymous London-based collector.
It is believed that there are only 11 surviving copies from that year.