More than 3,000 of her supporters chanted messages of solidarity as the crowd heaped flowers and nailed up hand-written greetings messages on heart-shaped cards outside the jail on Sunday to mark her 51st birthday.
Many, bearing flags with the slogan "We will overcome!", chanted her name.
Tymoshenko, who was twice prime minister, has been held at the Lukyanivska detention centre in Kiev since she was jailed this summer for seven years for abuse of office.
She and her supporters say the case is a political vendetta against her by President Viktor Yanukovich, who narrowly beat her in a bitterly fought run-off for the job in February 2010.
The affair has derailed Ukraine's plans for closer ties with the European Union.
The 27-member bloc says the trial was politically motivated and has called on Yanukovich to secure Tymoshenko’s release.
But he has refused to intervene and prosecutors are pressing ahead with investigations into a variety of fresh criminal charges against her.
"Yanukovich locked her up on purpose because he knows that she is worse than a nuclear war for him. If she gets out, he will not be president much longer," Viktor Redzhuk, a pensioner from the town of Zhitomyr, said.
Tymoshenko's daughter, Yevhenia, said outside the prison: "For me it's obviously not a celebration. Of course it's a holiday, because my mom was born, but for me it's a sad day, because she is in prison."
Police made no attempt to intervene or to disperse the crowd.
Supporters joined in when popular singer Nina Matvienko, one of several performers who Tymoshenko's Batkivshchyna party said would appear during the day, sang a Ukrainian folk song.
There was no sign of Tymoshenko at any of the windows of Luk'yanivska prison, a long, rose-coloured building set back from the road, and there was no way of knowing whether she could hear the crowds outside.
Supporters fear her health is declining and there have been reports that she is suffering from a recurring back problem.