Marquis grew up in Walnut, Illinois. His brother David died in 1892 at the age of 20; his father James died in 1897. After graduating from Walnut High School in 1894, he attended Knox Academy, a now-defunct preparatory program run by Knox College, in 1896, but left after three months. From 1902 to 1907 he served on the editorial board of the Atlanta Journal where he wrote many editorials during the heated election between his publisher Hoke Smith and Future Pulitzer Prize winner, Clark Howell (Smith was the victor).

Marquis began work for the New York newspaper The Evening Sun in 1912 and edited for the next eleven years a daily column, "The Sun Dial". During 1922 he left The Evening Sun (shortened to The Sun in 1920) for the New York Tribune (renamed the New York Herald Tribune in 1924), where his daily column, "The Tower" (later "The Lantern") was a great success. He regularly contributed columns and short stories to the Saturday Evening Post, Collier's and American magazines and also appeared in Harper's, Scribner's, Golden Book, and Cosmopolitan.

Marquis's best-known creation was Archy, a fictional cockroach (developed as a character during 1916) who had been a free-verse poet in a previous life, and who supposedly left poems on Marquis's typewriter by jumping on the keys. Archy usually typed only lower-case letters, without punctuation, because he could not operate the shift key. His verses were a type of social satire, and were used by Marquis in his newspaper columns titled "archy and mehitabel"; mehitabel was an alley cat, occasional companion of archy and the subject of some of archy's verses. The archy and mehitabel pieces were illustrated by Cartoonist George Herriman, better known to posterity as the author of the newspaper comic Krazy Kat. Other characters developed by Marquis included Pete the Pup, Clarence the ghost, and an egomaniacal toad named Warty Bliggins.

In 1909, Marquis married Reina Melcher, with whom he had a son, Robert (1915–1921) and a daughter, Barbara (1918–1931). Reina died on December 2, 1923.

Marquis was the author of about 35 books. He co-wrote (or contributed posthumously) to the films The Sports Pages, Shinbone Alley, The Good Old Soak and Skippy. The 1926 film The Cruise of the Jasper B was supposedly based on his 1916 novel of the same name, although the plots have little in Common.

Three years later Marquis married the Actress Marjorie Potts Vonnegut, whose first husband, actor Walter Vonnegut, was a cousin of American author, Playwright and satirist Kurt Vonnegut Jr. She died in her sleep on October 25, 1936.

On August 23, 1943, the United States Navy christened a Liberty ship, the USS Don Marquis (IX-215), in his memory.