The vote in the duchy of 465,000 people resulted in a majority of 57 percent.
The country’s Prime Minister Jean-Claude Juncker had staked his job on the outcome of the referendum, urging the traditionally pro-EU nation to turn the tide of ‘no’ votes against the EU constitution.
France and the Netherlands both voted against the constitution in May and June, sending the union into crisis and causing a majority of EU diplomats to proclaim the treaty dead.
But that prognosis has been rejected by Luxembourg’s Foreign Minister.
“Europe is a bit sick, Europe caught a cold, but today Luxembourg has given us a good cup of tea with honey,” Jean Asselborn said.
“As Luxembourg said ‘Yes’, the process can go on… and we will see at the very end how to react to those countries having said ‘No’,” Mr Juncker said.
Mr Juncker said with Luxembourg’s backing, the charter had now been ratified by 13 countries – just over half of the union’s 25 member states.
The constitution must be approved by each of the EU’s 25 members in order to come into force.
Mr Juncker acknowledged that ultimately it will have to either go to a fresh vote in France and the Netherlands or be renegotiated.
However, last month the union agreed to allow an extended period for undecided nations to consider the future of the treaty.
Britain, which has recently taken up the EU’s presidency, has decided to put on hold its own referendum plans, saying that while Luxembourg’s result was welcomed “there should be a pause for reflection and that decisions about further ratification of this treaty are a matter for each member state to decide.”