The show follows the adventures of stepbrothers Phineas Flynn and Ferb Fletcher, who live in the town of Danville and are determined to pursue an active summer vacation and defeat boredom. Their sister, Candace, is obsessed with "busting" their schemes and ideas, and usually calls their mother to report the boys' activities. The boys' pet platypus, Perry, meanwhile acts as a secret agent for an all-animal government organization called the "O.W.C.A." (Organization Without a Cool Acronym), fighting the evil Dr. Heinz Doofenshmirtz.

Much of the series' humor relies on running gags used in every episode with slight variation. For example, almost every episode features an adult asking Phineas if he is not too young to be performing some complex activity, to which he responds "Yes, yes I am." Perry and Doofenshmirtz's confrontations almost always lead to the eventual destruction of whatever contraption Phineas and Ferb have built that day. Ferb is silent by convention, usually uttering only a few lines per episode. Other running gags include catchphrases, such as Phineas's line "Ferb, I know what we're gonna do today!" and Doofenshmirtz's "Curse you, Perry the Platypus!"

Aspects of the show's humor are aimed at adults, including its frequent pop-cultural references and occasional double-entendres. Creator Povenmire, having previously written for Family Guy, sought to create a show less raunchy but with the same use of comic timing, employing humorous blank stares, expressionless faces, and clever wording. Povenmire describes the show as a combination of Family Guy and Spongebob Squarepants. Jeff Marsh said the show was not created just for kids, but simply did not exclude them as an audience.