Prague mayor Zdenek Hrib, from the anti-establishment Pirate Party, cancelled a twinning agreement with Beijing in October in protest at Chinese insistence on a one-China policy.
Hrib hailed the new twinning with Taipei as "most beneficial" for both parties on Monday, citing "shared democratic values, respect for fundamental human rights and cultural freedoms."
Taiwan has been ruled separately from China since the end of a civil war in 1949, but under its "One-China" policy, Beijing considers it a part of its territory, with reunification by force an option.
"Prague has its own choice to become a sister city with cities of the world and I think Beijing should also let Prague the right to choose," Taipei mayor Ko Wen-je told AFP after Monday's ceremony, speaking via an interpreter.
Hrib condemned China as an "unreliable partner" in an interview run by German newspaper Welt am Sonntag on Sunday.
He added China was "full of resentment" and was trying to influence Czech public opinion, and that he could not sign an agreement that forced Prague to "speak out against the independence of Tibet and Taiwan."
Monday's signature comes only days after Taiwan emphatically re-elected incumbent President Tsai Ing-wen, a result widely seen as a rebuff to China. Hrib also accused the Czech government of "neglecting" ideals of the peaceful 1989 Velvet Revolution that ended four decades of communist rule in former Czechoslovakia, as it bows to China on many fronts.