Leave campaigner Arron Banks is being investigated for "suspected criminal offences" over £8 million of campaign funding during the Brexit referendum.
The National Crime Agency probe was launched after the elections watchdog said it had reasonable grounds to suspect that Mr Banks was not the true source of the cash.
Mr Banks, one of the founders of the Leave.EU campaign, said he was confident the investigation would "put an end to the ludicrous allegations" against him.
The Electoral Commission also referred Leave.EU, its chief executive, Elizabeth Bilney, and the organisation that ran it, Better for the Country, to the NCA after carrying out a review.
Bob Posner, the Commission's director of political finance, said: "We have reasonable grounds to suspect money given to Better for the Country came from impermissible sources and that Mr Banks and Ms Bilney, the responsible person for Leave.EU, knowingly concealed the true circumstances under which this money was provided.
"This is significant because at least £2.9 million of this money was used to fund referendum spending and donations during the regulated period of the EU referendum."
Mr Banks said the Commission had referred him to the NCA under "intense political pressure" from anti-Brexit supporters.
He said: "I am confident that a full and frank investigation will finally put an end to the ludicrous allegations levelled against me and my colleagues.
"There is no evidence of any wrongdoing from the companies I own. I am a UK taxpayer and I have never received any foreign donations. The Electoral Commission has produced no evidence to the contrary."
Ms Bilney also accused the Commission of having a "biased approach" in its investigations and said she was confident she would be "exonerated".
She told BBC Radio 4's World At One programme: "I hope that the matter will be shortly concluded to demonstrate that no crimes have been committed."
"They are looking at it how they want to through their own biased lens," she added.
Mr Banks was criticised by the Commons Digital, Culture, Media and Sport Committee earlier this year for seeming to "want to hide the extent of his contacts with Russia".
But Ms Bilney dismissed suggestions that some of the money may have come from the Federation.
"I run the group of companies where the money was from and we don't have any transactions that are from Russia," she said.
"I completely comfortable that we have done everything above board."
NCA initiates investigation following Electoral Commission referral pic.twitter.com/qSzZfEWSEH
— NationalCrimeAgency (@NCA_UK) November 1, 2018
The Electoral Commission's review of referendum finances focused on £2 million reported to have been loaned to Better for the Country by Mr Banks and his insurance companies and a £6 million donation he made alone.
It said that, as well as having reasonable grounds to suspect he was not the true source of the cash, loans involved a company, Rock Holdings, based on the Isle of Man, which was impermissible under the rules.
The watchdog said it suspected Mr Banks, Ms Bilney and others involved in Better for the Country and Leave.EU concealed the true details of the financial transactions.
It believes a number of criminal offences may have been committed.
The NCA said its investigation relates to suspected electoral law offences.
A spokesman added: "While electoral law offences would not routinely fall within the NCA's remit, the nature of the necessary inquiries and the potential for offences to have been committed other than under electoral law lead us to consider an NCA investigation appropriate in this instance."