The middle aged man placed the bet via a banker's draft at odds of 1/4 when he visited a William Hill's betting shop in south-west London. He stands to make a profit of £100,000 if correct.
The bookies said it was believed to be the largest political bet ever in the UK, or even the world.
Few details have emerged of the punter, other than that he's from Surrey and does not have a Scottish accent.
Hill's spokesman Graham Sharpe said: "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."
Hill's have now shortened their odds on a No vote from 1/4 to 1/5, and are offering 10/3, out from 11/4, on a Yes vote.
Mr Sharpe said: "We are now well on the way to turning over £1million on the outcome of the Scottish Referendum which is exceeding our expectations."