The West calls Tymoshenko's seven-year sentence for abuse of office politically motivated and has refused to sign a landmark association agreement with Kyiv at a summit next week unless she is freed.
But members of President Viktor Yanukovych's Party of Regions on Thursday did not vote in favour of even one of the six bills meant to allow Tymoshenko's release, despite the presence of two top EU envoys, who have been lobbying for a solution for months.
Opposition lawmakers responded with chants of "Shame! Shame!" and urged Yanukovych to pardon Tymoshenko through a presidential decree.
"It is President Viktor Yanukovych who is personally blocking Ukraine's movement toward the European Union," opposition leader Arseniy Yatsenyuk told parliament in an emotional speech after the vote failed.
The Thursday vote was an opportunity for Ukraine to fulfil the main EU condition for the deal to be signed. But theoretically Yanukovych still has until right before the summit begins on Nov. 28 to secure Tymoshenko's release either by pardoning her or by having parliament pass the necessary law next week.