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The Tokyo High Court turned down the appeal filed by Masami Tsuchiya, 41, a court spokeswoman said on condition of anonymity, citing court policy. She refused to offer any other details.
Tsuchiya in January 2004 became the 11th member of the Aum Shinrikyo cult to be sentenced to death for the attack.
Prosecutors at the time said Tsuchiya's responsibility in the killings was second only to that of the group's guru, Shoko Asahara.
Tsuchiya, charged with murder and attempted murder in the subway gassing and other attacks, was accused of heading the group's drive to develop an arsenal of chemical weapons, including VX, mustard and sarin gases intended to trigger Armageddon.
Sarin was used in the March 1995 attack, in which cult followers punctured plastic bags of the gas on rush-hour subway trains. In addition to the deaths, the attack sickened some 5,000 people and shocked the country.
The proceedings against Tsuchiya and other senior leaders had dragged on for years, in part because they face a long list of charges involving masses of evidence and testimony. Tsuchiya fired his lawyers twice, delaying his case even further.
Asahara, the nearly blind self-styled messiah who once claimed more than 10,000 followers, was convicted and sentenced to die in April 2004.
Lawyers for Asahara, the religious name assumed by Chizuo Matsumoto when he founded Aum, petitioned Japan's Supreme Court this June in what could be their last chance to save him. A decision is expected at any time.
Raids of cult headquarters and confessions of leading members later revealed the cult had numerous plots to overthrow the government and operated labs to develop chemical and biological weapons.
The group has renamed itself Aleph and authorities say about 1,650 people in Japan and 300 in Russia continue to believe in Asahara's teachings.