Euphues is the protagonist and titular character of Euphues: The Anatomy of Wit. His name is derived from Greek meaning "graceful, witty." Euphues is indeed witty, and is also blessed with beauty and great fortune. Unfortunately, he often uses his many gifts for ill, choosing to chase hedonistic pleasure rather than uphold virtue.

Philautus is Euphues' friend. He is intelligent and kind, though his trust in Euphues turns out to have been misplaced when Euphues steals from him the affections of Lucilla, and attractive but fickle young woman whom Philautus had loved. Euphues does not have the last laugh, however. Lucilla once again changes her affections, this time for Curio, a man of "little wealth and less wit."

Other characters include Don Ferardo, a wealthy nobleman and Lucilla's father, and Eubulus, a wise old man saddened by Euphues' wicked use of his many gifts.