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The middle-aged man, who has not been named, spoke with an English accent as he placed the bet in the prosperous southern county of Surrey near London at odds of 1/4 on, meaning he will make £100,000 pounds if he wins, said bookmakers William Hill.
"As far as we can ascertain this is the biggest political bet ever struck," said Graham Sharpe, media projects director at William Hill.
The bet is twice the £200,000, also put on a "no" vote, staked by a Scottish man earlier in the independence campaign.
By way of comparison, a customer similarly gambled £200,000 on David Cameron, now Britain's prime minister, winning the leadership of his Conservative Party in 2005.
Scots will vote in a referendum on September 18 on whether to leave the United Kingdom. Polls currently suggest roughly 40 percent of Scots will vote against independence and 30 percent will vote for, but many voters remain undecided.
William Hill believe they will turn over more than £1 million in bets on the independence outcome.
The bookmakers said they had shortened their odds on a "no" vote to 1/5, suggesting this was now more likely to happen than previously thought. Their odds on a "yes" vote stand at 10/3, meaning winners getting £10 back for every £3 they bet.