The Ecumenical Patriarch of Constantinople Bartholomew I signed on Saturday the decree of independence which formalised a split that has been brewing for months.
The Ukrainian church has formally been under the Russian Orthodox Church for centuries.
The Russian government and the Russian Orthodox Church strongly oppose the move.
The Russian Orthodox Church has severed ties with Istanbul and suggested they will no longer attend gatherings by the Ecumenical Patriarchate of Constantinople - the nominal head of the Eastern Orthodox Churches.
Bartholomew I said in his address at the Patriarchal Church of St George in Istanbul: "The pious Ukrainian people have awaited this blessed day for seven entire centuries."
Bartholomew I first approved the Ukrainian church's request for autocephaly, or independence, last October.
Epiphanius I of the Orthodox Church of #Ukraine arrives in Istanbul to receive the Tomos or letter of autonomy from the Patriarch of Constantinople. This break from Moscow will enrage #Russia and flare up tensions in orthodox world. pic.twitter.com/mlGGfpqwok
— Melvyn Ingleby (@MelvynIngleby) January 5, 2019
Many Ukrainians wanted their church to separate from Russia over fears of political influence from the Kremlin.
Ukraine's President Petro Poroshenko made the creation of the independent church a part of his campaign platform for the upcoming election in March.
He also attended the signing.
The split will likely cause the Russian Orthodox Church to lose 30 to 40 per cent of its 150 million members, The New York Times has reported.
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