The characters were represented as black face minstrel show caricatures of African Americans. Gosden and Correll were white actors who came up through the minstrel tradition and were highly skilled voice actors. In the early days of the program they played all the male character roles; between the two, they voiced over 170 distinct characterizations in the show's first decade.

The title characters, Amos Jones and Andrew H. "Andy" Brown, were depicted as being under-educated blacks from rural Georgia going North to find work in the big city of Chicago.  Amos was naïve but honest, hard-working, and a dedicated family man. Andy was more blustering, with overinflated self-confidence. After a lack of success finding work, they started the Fresh Air Taxi Company, so called because it's only cab had no windshield.  Andy was a dreamer and tended to let Amos do most of the work. Other regular characters included their lodge leader George "The Kingfish" Stevens, who was always trying to lure the title characters into get-rich-quick schemes, and "Lightning", a slow-moving Stepin Fetchit-type character. Kingfish's catchphrase "Holy mackeral!" quickly entered the American lexicon.

Amos & Andy was one of the earliest success stories of radio syndication, and many stations besides WMAQ carried the program using prerecorded records. In August 1929 Gosden & Correll moved the show to NBC which offered them more money (the first NBC broadcast was on August 19). At the same time the story line of Amos & Andy had the title characters move from Chicago to Harlem, New York City, where they were soon joined by the rest of the regular characters.