Born at Tsarskoie Selo, his father was Czar Alexander II Nicholaievitch, and his mother Marie Princess von Hesse-Darmstadt. He was the brother of Czar Alexander III Aleksandrovich, and the favourite uncle of Czar Nicolas II.

Between 1891 and 1905, he was appointed by Nicolas II as Governor General of Moscow. A radical conservative, his policies made him a polarizing figure. In 1892, as the governor of Moscow, he executed the state's order and expelled the large portion of the Jewish population from the city. The Khodynka Field tragedy, in 1896, tarnished his reputation.

He was on of the Seven Homosexual Grand Dukes. His sexuality conflicted with his intense religious beliefs and the expectations of his position. He married Princess Elizabeth of Hesse. Contrary to his belief, the marriage was happy, in its own way. Unusual for royal couples, they slept in the same bed for all of their married life. Forced to defend Sergei against rumors of discord, Elizabeth was devoted to her husband and treasured his memory after his death.

Highly controversial, he was targeted by revolutionaries and was assassinated by a terrorist bomb at the Kremlin, in Moskow, thrown at him by Socialist-Revolutionary terrorist Ivan Kalyaev.

Sergei Aleksandrovich was buried in the Kremlin, but his grave remained unmarked beneath a Car park the Bolsheviks later built in the Kremlin.

After the fall of the Soviet government, he was exhumed and reburied with due pomp and ceremony in the Romanov family vault in the Kremlin, with the living descendants in attendance.