A 421-273 vote of MEPs was hailed as a "triumph" as MEPs cheered the result - but months of negotiations with EU governments and the European Commission now follow.
Most British Labour MEPs backed the call to end the opt-out - clashing with Prime Minister Gordon Brown, who wants it to stay.
At least 14 other EU countries now take advantage of an opt-out, once championed only by Britain as vital to business flexibility.
With an economic downturn in full swing, more countries want the option of exceeding the 48-hour week - and earlier this week the Commission admitted it had reversed its own opposition to keeping the opt-out because of pressure from so many member states.
But today MEPs bucked the trend, voting by a bigger margin than expected in favour of a resolution that the opt-out should be gone by 2011.
It would force employers across Europe to stick to a maximum 48-hour week, averaged over a year.
The arrangement to average the maximum out provides sufficient short-term scope for bosses and workers to work longer hours if necessary, supporters of the ban argued.
But opponents said it should not be up to the EU to determine the working patterns of very different employment cultures in the member states.
Tory MEP Philip Bushill-Matthews said after the vote: "Socialist MEPs have won the battle today, but they must not be allowed to win the war.
"The British Government must dig in and defend the opt-out. Fifteen EU nations now take advantage of the flexibility provided by the opt-out and none of them should back down."
He went on: "It should never be the place of the European Parliament to tell people they cannot work - particularly during a downturn. Scrapping our working time opt-out is even more nonsensical in today's economic climate than ever before.
"This is a double failure of Gordon Brown. Not only has he failed to control his MEPs, but he also naively signed up to a package deal that saw Britain give ground on the Agency Workers Directive in exchange for our working time opt-out.
"His folly was to assume the Left in the European Parliament would not sabotage the deal. British businesses have been given two damaging pieces of employment legislation for the price of one."
Fellow Tory MEP Daniel Hannan claimed that today's decision, if upheld after the next stage of three-way "conciliation", would cost the UK economy between £47.4bn and £66.45bn by 2020 - costing the average family more than £1,000.
"Quite apart from being unaffordable, this is morally wrong. If I want to work for you, and you want to employ me, and both of us are happy with the terms of our contract, then it is quite wrong for the state - let alone the EU - to come between us and declare that contract illegal."
Liberal Democrat MEP Liz Lynne attacked Labour MEPs who went against Mr Brown.
"The UK Government should have been able to rely on all their own MEPs to support them in the retention of the opt-out, but that was not the case," she said.
"For months now the Government has had to work with me on this as it couldn't get the support of its own party."
The outcome was a "bitter blow" for businesses and workers, she added.
"The opt-out should be retained, as long as it is truly voluntary. Workers should be allowed to earn overtime if they wish. Scrapping the opt-out would push people into illegal work where they would not be covered by health and safety legislation.
"Let us hope that when we go into discussions with the Council of Ministers on this that they will not give in to the Parliament's view, and maintain the opt-out."
The British Chambers of Commerce (BBCC) condemned the outcome as "foolish".
BBCC director-general David Frost, speaking just before the vote, said: "MEPs would be foolish to try and end the UK's working time opt-out, especially during a painful recession.
"The opt-out provides the essential flexibility that many businesses and their employees are relying on to get through this downturn."
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