It says the allegations relate to 20 people being paid from the EU budget as assistants to the French party's MEPs.
There are doubts whether they do work directly connected to the parliament.
The party leader said she would file a complaint against the parliament's president over the "false accusations".
Ms Le Pen also said French Prime Minister Manuel Valls was "mobilising his socialist friends against the National Front".
Mr Valls has said he fears for his country over the rise of the Front, which is leading in some French opinion polls.
He warned that the FN could win the next presidential election in 2017, saying its policies were a "disaster" for France.
In a statement, the European Parliament said its president, Martin Schulz, had alerted the EU anti-fraud agency Olaf about information noted in February relating to the salaries of FN staff members.
It said many of the assistants had listed their working address as the National Front's headquarters in Nanterre, in the western suburbs of Paris, while some had different job descriptions on their EU contracts compared with FN documents.
It added that party paperwork also showed certain assistants did not work for the MEPs listed on their EU contracts.
Ms Le Pen denounced the move as "political manipulation", according to AFP news agency.
"The EU parliament's president is bringing out the big guns," she wrote on Twitter.
"A complaint will be filed against him [Martin Schulz] for making false allegations," she added, although she did not say where such an action would be brought.
A spokesperson for Olaf said it was assessing the information to decide whether there were sufficient grounds to launch an official investigation.
The FN won France's European Parliament elections in May 2014 with 25% of the vote, taking 24 seats.
The European Parliament has called in the EU's anti-fraud squad to investigate possible financial irregularities involving Marine Le Pen's far-right National Front (FN).