Because of congenital cataracts, Vierne was nearly blind at the time of his birth. In his youth, he was able to undergo an operation that partially restored his eyesight.

In 1889, Vierne became a pupil of César Franck at The Paris Conservatory. Franck died the next year from complications of an automobile accident. Vierne's father also died the same year. Charles-Marie Widor succeeded Franck in becoming Vierne's teacher.

Vierne became an assistant to Widor at the church of Saint-Sulpice in Paris from 1892 until he became principal organist at The Cathedral of Notre-Dame de Paris in 1900. He held this post until his death in 1937, just a few months after the death of Widor.

Vierne also taught at The Paris Conservatory first as Widor's assistant in 1894-96 and then as the assistant of fellow composer Alexander Guilmant (1837-1911). After 1911 he taught at the Schola Cantorum.

After the outbreak of World War I, Vierne suffered an outbreak of glaucoma, which caused him once again to lose his sight.  Because of this, he took a 4-year leave of absence from his post at The Cathedral of Notre-Dame. Vierne's pupil, Marcel Dupre, took over during his absence. When Vierne returned to Paris, he found the organ at Notre-Dame had suffered extensive war damage. Wanting to raise money for repairs, he began a concert tour that brought him to the United States in 1927, playing concerts on the famous Wanamaker Grand Court Organ in Philadelphia, Pennsylvania.

Widor died at the console of this organ while performing in concert at The Cathedral of Notre-Dame of what may have been a heart attack. His student Maurice Duruflé was with him.