Turkey also issued a declaration making clear the signing does not mean Ankara now recognises Cyprus. Turkey recognises only a breakaway Turkish Cypriot enclave on the island.
Because of political sensitivities, there was no signing ceremony. British EU ambassador John Grant signed the text on behalf of the 25-nation bloc and sent it to Turkish ambassador to the EU Oguz Demiralp.
British and Turkish officials said in advance of Friday's signing they expected the wording of the Turkish declaration would not pose new legal problems that could obstruct the historic launch of accession negotiations on 3 October.
However, Cyprus said it would be "unthinkable" for Turkey to refuse to recognise an EU member state while at the same time seeking the agreement of all EU countries to open talks.
The EU must still approve unanimously a negotiating mandate for the talks, giving Nicosia a potential veto. Cyprus, lying just south of Turkey, was partitioned in a Turkish invasion in 1974 in response to a Greek Cypriot coup.
Turkey met the EU's other condition for starting talks in April, when it brought into force six crucial packages of legislative reforms.
The executive European Commission determined last year that Turkey had sufficiently met the EU's political criteria on democracy, human and minority rights and the rule of law to start accession negotiations.
But it said the talks would probably take at least a decade and Turkey could not join before 2014, when a new long-term EU budget comes into force.
A pan-European opinion survey published by the Commission this month showed 52% of EU citizens opposed membership for the mainly Muslim country and only 35% supported it. Hostility was strongest in Austria, Germany and France.