"This is a great day for Europe... the European Union stands by your side today more than ever before," he emphasized.
In remarks before the signing, the president of the European Council, Herman Van Rompuy, also remembered those who died in Ukraine.
"In Kiev and elsewhere, people gave their lives for this closer link to the European Union," he said. "We will not forget this."
Van Rompuy said that closer political and economic ties would bring greater security, stability and prosperity to Ukraine and the two other former Soviet states also signing EU Association Agreements on Friday, Georgia and Moldova.
"Our joint goal is your full integration into the market of the European Union," Van Rompuy told the three leaders, adding that they must push forward with necessary reforms.
He also expressed the solidarity of the European Union with the three nations in the face of Ukraine's "very difficult" security situation, and the "uncertainties" looming over Georgia and Moldova's relationships with Russia.
At the same time, he stressed that Russia had nothing to fear from those nations' closer ties with Europe, adding that the European Union would engage with Russia to work for peace.