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Secret Squirrel (voiced by Mel Blanc) serves as a secret agent, taking orders from his superior Double-Q (voiced by Paul Frees) of the International Sneaky Service. His designation is Agent 000. Secret Squirrel is assisted in his adventures by fez-wearing sidekick Morocco Mole (also voiced by Paul Frees impersonating Peter Lorre). Morocco Mole has a thick Middle Eastern accent and likely has Asperger's syndrome. The pair fights crime and evil enemy agents using cunning and a variety of spy gadgets, including a machine gun cane, a collection of guns kept inside Secret's trench coat, and a variety of devices concealed in his fedora (which he almost never removes).

Secret Squirrel's recurring arch-enemy is Yellow Pinkie (also voiced by Frees), a parody of both Auric Goldfinger from Goldfinger and Kasper Gutman from The Maltese Falcon. The last episodes introduced Hy-Spy (again voiced by Frees), the master of scientific criminology