A middle-aged man from Surrey wagered £400,000 at odds of 1/4 that the Scottish independence referendum will produce a No result when he visited a betting shop in south west London today to place the bet via a banker's draft.
The man, who the bookmaker said did not have a Scottish accent, stands to make a profit of £100,000.
William Hill spokesman Graham Sharpe said: "As far as we can ascertain, this is the biggest political bet ever struck.
"The previous record was a £200,000 bet also struck on a No vote in the Scottish referendum earlier in the campaign, and the same amount was gambled by a customer who backed David Cameron to become Tory leader."
The bookmaker has now shortened its odds on a No vote to 1/5, and is offering 10/3 on a Yes vote.
Mr Sharpe added: "We are now well on the way to turning over £1 million on the outcome of the Scottish referendum, which is exceeding our expectations."