It remains unclear whether the deputies who stormed out on Wednesday have permanently quit their positions, but the move is nonetheless seen as bringing the country's shaky coalition one step closer to collapse.
"The people of Serbia should know that the parliament as a legitimate institution no longer exists due to the illegal revoking of mandates from the deputies of the Democratic Party of Serbia (DSS)," said the party's deputy president, Dragan Marsicanin.
Serbia's pro-Western Prime Minister, Zoran Djindjic, has been involved in an mounting power struggle with President Kostunica, a moderate nationalist, in recent months.
It was Mr Djindjic's allies in parliament who expelled the 21 DSS deputies on Tuesday.
Fifty DSS deputies were also sacked two weeks ago for failing to attend parliamentary debates.
Their repeated absences meant that debates on key issues could not take place, a political tactic which Mr Kostunica has defended.
Mr Djindjic and Mr Kostunica and their respective parties joined forces in 2000 to oust former president Slobodan Milosevic, but have clashed over a range of issues, including economic and legal reforms, and co-operation with the international war crimes tribunal in The Hague.
Early elections
Last week, Mr Kostunica's Democratic Party of Serbia (DSS) formed a shadow government which aides said would start to "monitor" the work of Mr Djindjic's cabinet, which DOS ministers quit last year.
Analysts said this effectively amounted to their joining the opposition, and that the DSS was seeking to form a clear identity for itself before parliamentary elections - due at the start of next year, but likely to be held early.
Mr Kostunica is particularly keen on the idea of early elections.
Mr Djindjic has said early elections are possible if the parliament remains unable to function due to the lack of a quorum.
But correspondents say early elections would certainly not be in the interests of Mr Djindjic or his party, which has been lagging in the polls.
Mr Djindjic has made clear on several occasions that he feels aggrieved at having to bear responsibility for unpopular, but unavoidable decisions which Mr Kostunica is able to distance himself from.
The prime minister has accused the president of trying to score "patriotic points" with his opposition to the extradition of war crimes suspects, while knowing that the government has no option if it wishes to continue receiving vital financial aid.