The decision came one day after Tymoshenko's attorneys argued before the European Court for Human Rights that the former leader was held illegally and under inhumane conditions before her trial, Voice of America reported.
Tymoshenko is serving a seven-year prison sentence in connection with a natural gas deal she brokered with Russia in 2009 while serving as prime minister.
In turning down her appeal, the high court said she had no grounds, the Russian news agency RIA Novosti reported.
Tymoshenko's lawyers argued that the criminal case against her lacked evidence.
Some 200 supporters of Tymoshenko, chanting "Freedom to Yulia," gathered outside the courthouse before the verdict was announced, the Ukraine news agency UNIAN reported.
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