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While loosely based on Sousa's autobiography Marching Along, the film takes considerable liberties and dramatic license, often expanding and extrapolating on themes and passages in the book.
In the 1890s, Sergeant Major John Philip Sousa, the director of the United States Marine Band, leaves the Marine Corps after his enlistment expires to form his own band because he is not paid enough to provide for his wife Jennie (Ruth Hussey) and their children. As a favor for his splendid service, he is allowed to take along Private Willie Little (Robert Wagner), who is credited with designing the Sousaphone and naming it after his mentor (in real life, Sousa himself designed the instrument).
Willie persuades Sousa to go with him to a "concert" where some of Sousa's songs will be performed. In fact, it is a music hall, where Willie's girlfriend, Lily Becker (Debra Paget), is one of the performers. When the police raid the place for indecency (by 1890s standards), the trio barely manage to get away.
Sousa forms his band and is very successful. A mention in the book that Sousa discouraged the married men in the band from bringing their wives on tour is expanded into a subplot where Willie and Lily get married in secret so they can continue touring together with Sousa. An episode shows Sousa's Band playing at the Atlanta, Georgia Cotton States and International Exposition (1895), despite the attempt by Colonel Randolph (Finlay Currie) to cancel their contract. Sousa has his musicians play "Dixie" as they march up, putting the crowd in a cheerful mood. A listing of his song list for the performances includes "Dixie" as every second one. The crowd cheers, and Randolph welcomes Sousa. This stays relatively close to fact.
Sousa tours the world, and is honored by the crowned heads of Europe. Late one night, he spots Willie sneaking into Lily's train compartment late; Sousa's wife has to defuse his indignation by letting him into the secret.
When the USS Maine is sunk by a suspicious explosion, precipitating the Spanish-American War, both Willie and Sousa reenlist. However, Sousa is kept from the actual fighting and instead sent on a sea voyage by an outbreak of typhoid. The inspiration for the title march is depicted with a voiceover of Webb quoting Sousa's actual description of the event while at sea (however, the sea voyage in real life was due to Sousa and his wife rushing back to the U.S. from a vacation in Europe upon the sudden death of his manager).
During a rehearsal of El Capitan, starring Lily, the Sousas receive a letter from Willie in which he reveals he was wounded in the knee in a friendly fire incident in Cuba. His leg has to be amputated. After the war, while recovering at the Brooklyn Navy Yard Hospital, he is called upon to rejoin Sousa's band in a surprise concert, where the band plays the title march in public for the first time. (In real life, the march was first played publicly at the Academy of Music in Philadelphia on May 14, 1897, much earlier than depicted.) The film then cuts away to modern Washington, D.C., where the ghostly spirit of Sousa leads a real marching band.