The EU demanded that Ukraine boost the rule of law and free the jailed former prime minister before the two could sign a landmark political and free trade deal.
But last week President Viktor Yanukovych's government stunned the West by halting all negotiations on the deal that would have set it on a path to EU membership, saying it wanted better terms.
Yanukovych said he would still travel to the two-day summit in Vilnius to continue negotiations but his government's shock decision sparked the biggest rallies since the pro-democracy Orange Revolution in 2004.
"If Yanukovych takes a positive decision, I passionately ask you to sign the agreement on Friday without any hesitation and conditions including those that are related to my release," Tymoshenko, 53, said in a message read out by her daughter Yevgenia late Wednesday.
"I thank you for steadfastly defending democracy in Ukraine. But today it's necessary to release not just separate political prisoners," said Tymoshenko who on Monday launched a hunger strike in solidarity with pro-European protesters.
"It's necessary to free Ukraine. That means it's necessary to sign the agreement if Yanukovych agrees to it."
"By signing the agreement with him, you would help an entire nation to overcome a civilisational abyss created by erroneous ideologies and agressive empires, you would make another important step towards re-uniting the entire Europe."
The Ukrainian government's decision to scrap the EU pact has brought tens of thousands onto the streets since Sunday.
Setting up camp in central Kiev and led by opposition leaders including world boxing champion Vitali Klitschko, protesters has staged mass rallies since Sunday.
Yanukovych has called for calm after the protests turned violent, with riot police firing tear gas and protesters hurling traffic cones and rocks at the security forces.
'Offer stays on the table'
Yanukovych stressed he would wait for better EU terms including financial aid before considering signing the deal. He said the bloc offered insufficient compensation for the damages Ukraine would suffer by rupturing ties with historic master Russia.
The EU said it was still ready to talk about the deal but the ball was in Ukraine's court.
"Our offer stays on the table," said the EU's Enlargement Commissioner Stefan Fuele.
The debate over Ukraine's future has turned into a heated diplomatic tussle between the EU and the Kremlin.
Russia wants its smaller neighbour to join a Moscow-led Customs Union which already includes the ex-Soviet states of Belarus and Kazakhstan. Moscow has repeatedly threatened Ukraine, which heavily depends on Russian natural gas, with economic retaliation if it signed the EU pact.
Top EU officials have said they "strongly disapprove" of Russian pressure on Kiev.
Russian President Vladimir Putin has responded by advising "our friends in Brussels, my personal good friends in the European Commission, to hold back on the sharp words".
Kiev's move to scrap the deal with the EU came after parliament failed to adopt legislation that would have freed Tymoshenko.
Shortly after she lost to Yanukovych in a 2010 presidential election, she was sentenced to seven years on abuse of power charges that the West sees as politically motivated.
Ukrainian opposition leaders Klitschko, Arseniy Yatsenyuk and Oleg Tyagnybok are also expected at the Vilnius summit.