Dvorak was born in Nelahozeves near Prague where he spent most of his life. He studied music in Prague's Organ School at the end of the 1850s, and through the 1860s played viola in the Bohemian Provisional Theatre Orchestra which was from 1866 conducted by Bedoich Smetana.

From 1892 to 1895, Dvorak was director of the National Conservatory in New York City. The Conservatory was founded by a wealthy socialite, Jeannette Thurber, who wanted a well-known composer as director in order to lend prestige to her institution. She wrote to Dvorak, asking him to accept the position, and he agreed, providing that she were willing to meet his conditions: that talented Native American and African-American students, who could not afford the tuition, must be admitted for free. She agreed to his conditions, and he sailed to America.

It was during his time as director of the Conservatory that Dvorak formed a friendship with Harry Burleigh, who became an important African-American composer. Dvorak taught Burleigh composition, and in return, Burleigh spent hours on end singing traditional American Spirituals to Dvorak. Burleigh went on to compose settings of these Spirituals which compare favorably with European classical composition.

It was during his visit to the United States that he wrote his most popular work, the Symphony No.9 'From the New World'.

Also while in the USA he heard a performance of a cello concerto by the composer Victor Herbert. He was so excited by the possibilities of the cello and orchestra combination displayed in this concerto that he wrote a cello concerto of his own, the Cello Concerto in B minor (1895). Since then the concerto he wrote has grown in popularity and today it is frequently performed. He also left an unfinished work, the Cello Concerto in A major (1865), which was completed and orchestrated by the German composer Gunter Raphael between 1925 and 1929.

Dvorak was a colorful personality. In addition to music, there were two particular passions in his life: locomotive engines, and the breeding of pigeons.

He eventually returned to Prague where he was director of the conservatoire from 1901 until his death in 1904. He was interred in the Vysehrad cemetery in Prague.