It voted to dismiss the central election commission and refused to grant it funds for early elections.
In an extraordinary session broadcast live on television the assembly voted 450 to 261 in favour of a resolution calling for “an interruption of the powers of the central electoral commission’s members”.
The president had earlier arranged a meeting with parliamentary leaders after accusing Viktor Yanukovich, the prime minister and his chief rival, of attempting to expand the parliamentary majority by illegally recruiting MPs loyal to him.
The Interfax news agency quoted a source in the Kremlin as saying that Yushchenko had asked Vladimir Putin, the Russian president, to postpone talks between the two men scheduled for Tuesday in Moscow.
Yanukovich said the president could suspend the decree or simply not publish it to allow “the country to carry on and develop in calm, civilised fashion”.
Anatoly Hrytsenko, the defence minister and one of a tiny minority of cabinet members allied to Yushchenko, said he and the armed forces would obey orders only from the president.
Yushchenko swept to power on the back of the “Orange Revolution” in 2004, a popular movement that rejected Russian interference in Ukrainian politics.
But since the victory he has seen his power-base eroded by Yanukovich, the man he defeated in 2004.
Yushchenko has clashed with the prime minister since appointing him in August, threatening to dissolve the assembly unless the three-party governing coalition stopped recruiting individual opposition members to its ranks.
He appointed Yanukovich after his allies failed to form a government. The prime minister’s initial 239-strong coalition in the 450-seat assembly has been boosted by defections to 260.
Yanukovich is seeking 300 votes to be able to overturn presidential vetoes.
At the beginning of Monday’s talks Yushchenko threatened to dissolve parliament and call an election unless the coalition abandoned its “anti-constitutional” actions of persuading individual opposition members to join its ranks.
Even if all sides agree to take part, an election could produce a stalemate little different from the outcome of last year’s poll after which a coalition of “Orange” groups collapsed and Yanukovich took over as prime minister.
Some observers fear if ministers and parliament resist the poll, crowds both in favour and opposed to it could pour on to the streets, raising the possibility of confrontations.
Yushchenko’s dissolution of parliament will please the tens of thousands of protesters who rallied in Kiev over the weekend to back calls by opposition leader Yulia Tymoshenko, a former prime minister, for the president to dissolve parliament.
Tymoshenko was fired as Yushchenko’s first prime minister within eight months after her government split into competing factions.
She and other opposition leaders say new elections are needed as the expanded coalition no longer reflects how people voted in the previous election held last year.
Yanukovich’s Regions Party leads in opinion polls ahead of Tymoshenko’s bloc, with the president’s Our Ukraine lying third.