"We believe we are irreversibly on the road to EU membership," said Serbian Foreign Minister Vuk Jeremic.
Serbian nationalists, however, said they would rescind the agreement if they take power from EU backers in the May 11 election prompted by the Kosovo declaration of independence in February, The Times of London said.
Nationalist party members say the EU encouraged Kosovo to declare independence and said they would move closer to Russia if they are voted into power.
"We will never allow anybody to sign for the independence of Kosovo in the name of Serbia and that is why today's signature is not worth anything," said the nationalist Serbian Prime Minister Vojislav Kostunica.
It is unlikely the agreement would be embraced completely by EU officials until Serbia hands over fugitives Ratko Mladic and Radovan Karadzic to the International Criminal Tribunal for Yugoslavia. The two men are wanted for crimes against humanity for their involvement in the massacre at Srebrenica in 1995 that left more than 5,000 people dead.