Seymour was admitted to the New York state bar in 1832. He then served as military secretary to Governor William L. Marcy (1833–39), was a member of the New York Assembly (1842–46), and was elected mayor of Utica in 1842. Nominated for governor in 1850, he failed to win election but succeeded two years later. In 1854, however, he lost his bid for a second term, mainly because of his opposition to a bill prohibiting the sale of intoxicating liquor.

A conservative in national politics, Seymour advocated compromise to preserve the Union and avoid war. When the Civil War began he gave his full support to the Union cause, but he opposed President Lincoln's policies on emancipation, conscription, and military arrests. As governor of New York (1863–65) he felt that the draft quotas discriminated against New York City, and he appealed to Lincoln to remedy the situation. When draft riots occurred in the city in July 1863 he took measures to put down the disorders, but his conciliatory attitude toward the rioters weakened him politically.

After the war Seymour was active in the movement to rebuild the fragmented Democratic Party. In 1868 he was nominated as the Democratic candidate to run against General Ulysses S. Grant; although the popular vote was fairly close, Seymour received only 80 electoral votes to Grant's 214. Seymour retired to private life, but remained active in New York state politics.