Young Frank Blair Jr. was a bright but difficult student, who was expelled from several private schools and later from the University of North Carolina and Yale. In 1841 he managed to complete his studies at Princeton, but faculty objections to his rude behavior postponed his graduation until the following year. In 1842 he attended law school at his father's alma mater, Transylvania University in Lexington, Kentucky. After passing the bar, he joined his brother's law firm in St. Louis, then worked in the law office of Democratic Senator Thomas Hart Benton. In 1845 he traveled to the west in an effort to improve his health. At the commencement of the Mexican-American War, he enlisted in the army, then returned to St. Louis at the end of the war to resume his law practice and briefly edit the Missouri Democrat. He married Appoline Alexander; they had eight children.

Blair was a slaveowner, but opposed the expansion of slavery to the American West and favored the colonization of blacks to Africa. In 1848 he helped to found the Free-Soil party in Missouri and edited the party's organ, the Barnburner. His energetic condemnation of slavery and its expansion provoked an assassination attempt on his life. He did endorse, however, the Compromise of 1850 which allowed the electorates in the Utah and New Mexico territories to vote on the slavery issue, in exchange for California entering the Union as a free state. His stance on the 1850 Compromise led to a break with his political mentor, Senator Benton.

Blair represented the Democratic party first in the Missouri legislature (1852-1856), then in the U. S. Congress (1856-1858). He was the only Free-Soiler in Missouri's congressional delegation, and supported Republican John C. Frémont for president in 1856. His maiden speech on the House floor, partly written by his father, depicted slavery as a national problem, rather than as a regional institution. Arguing that slavery's dissolution was inevitable, he advocated gradual emancipation and the colonization of free and freed blacks to Africa. In 1859 Blair gained national recognition for an anti-slavery speech (another collaboration with his father), in which the congressman articulated his plan of gradual emancipation, colonization, and free labor. The oration was published as The Destiny of the Races on This Continent.

In 1858 Blair lost his bid for reelection to Congress as a Democrat. In 1860 he became a Republican and after endorsing Missouri's Edward Bates for the presidential nomination, switched to back Abraham Lincoln at the national convention. Blair campaigned vigorously for Lincoln in the general election and was himself elected by a narrow margin to Congress as a Republican. As Southern slave states seceded and the threat of civil war mounted, Blair personally financed the organization of paramilitary units (called "Home Guards") to defend St. Louis and Missouri for the Union. His home state was bitterly divided and became a bloody battleground during the Civil War. In the early months of the war, Blair led state militia units against Confederate troops before leaving to take his seat in Congress, where he chaired the House Committee on Military Defense. His actions in Missouri were instrumental in keeping that state within the Union.

Blair persuaded President Lincoln to name Frémont as Western Department commander, but the congressman soon became critical of what he considered to be Frémont's incompetence and lobbied for the general's removal. In 1862 Blair resigned from Congress to take a commission in the Union army, and returned to Missouri where he used his own money to raise seven infantry regiments. After the siege of Vicksburg, Mississippi (March 29-July 4, 1863), he was promoted to the rank of major general. He also saw action in the Chattanooga Campaign (October-November 1863) and in General William Tecumseh Sherman's March to the Sea (November-December 1864). In 1862 Blair was reelected to Congress as a Republican, so resigned from the army to take his seat in the House on March 4, 1865.

In Congress, Blair backed the more lenient Reconstruction plans proposed by President Lincoln and, after Lincoln's assassination, by President Andrew Johnson, while he fought strenuously against the policies of the Radical Republicans. He so embittered his congressional colleagues that they rejected President Johnson's nomination of him as revenue collector in St. Louis and as U.S. minister to Austria.

In 1868 the Democratic party nominated Blair as Horatio Seymour's vice-presidential running mate. During the campaign, Blair advocated nullification of the Reconstruction acts and predicted that if Ulysses S. Grant, the Republican presidential nominee, were elected, his presidency would degenerate into a military dictatorship. Blair's harsh words and abrasive personality tended to alienate potential supporters and provided fodder for his Republican opponents. He and Seymour lost the 1868 election.