The biggest country in the region, Romania has long been trying to carve out a role as a regional mediator before and after it joined the European Union in January 2007.
"Conditions to recognize the new entity are not fulfilled," parliament said in a declaration which got overwhelming backing by deputies.
"The parliament stresses that the decision in Pristina and the potential recognition by other states of the unilaterally declared independence cannot be interpreted as a precedent for other areas."
Romanian politicians have expressed opposition to granting independence to Serbia's breakaway province and have repeatedly said it may create a dangerous precedent in Europe and elsewhere.
Radical parties say Kosovo secessionist moves may serve as an example for Romania's ethnic Hungarians, living mostly in the central province of Transylvania, to try to break away.
The ethnic Hungarian party, UDMR, a member of the centrist government, was the country's only political grouping that has hailed Sunday's independence declaration.
"The birth of the new state has scattered Europe ... right here in the heart of it we have regions with similar demands. Just a spark is needed to make the relative balance explode," said Gandul newspaper editorialist Eliza Francu.