The Senate voted 54-25 to ratify the document just 75 days after it was approved by the lower house.
The treaty now goes to Czech President Vaclav Klaus, who opposed the treaty, saying it would hurt Czech sovereignty. However, it is not clear if he has the constitutional right to refuse to sign it, Prague Radio said.
The Lisbon treaty needs ratification by all 27 EU member states before taking effect. Ireland, Germany and Poland have yet to ratify it.