Bookmakers said the frenzy of excitement over the looming vote has prompted some people to place bets for the first time.
They include a woman from central London who has placed her first ever bet by putting £100,000 on the UK voting to stay. She stands to make £40,000 if the vote goes her way.
And another woman from Kingston in south-west London has gambled for the first time by staking £10,000 on Leave, according to William Hill.
Betfair says £40.5m has already been traded on the Betfair Exchange on the referendum — with £20.25m staked by backers and £20.25m put up by layers.
This means it has beaten the Scottish independence referendum to be the most gambled upon political event in British history.
While William Hill spokesman Graham Sharpe said he is expecting a massive spike in betting on Thursday as most people wait until the last minute to decide to gamble.
“We are expecting to see a big flurry of betting on Thursday, that’s what happened in the Scottish independence referendum.”
“There is a growing appetite for betting in politics, the Scottish independence referendum showed us that. We saw a similar phenomenon in the general election, the Labour leadership, and the London mayor.
“It has spread from a niche market with people in the Westminster bubble the main clients seeking bets, now there is much more interest, people feel much more connected to this event.”
William Hill has cut their odds for Remain twice, as they went from 4/11 to 1/3 and then to 2/7 (a 77% chance), while Leave was lengthened twice, from 11/5 to 9/4, then to 13/5 (a 27% chance).
While Betfair also placed the odds for Remain at 2/ — the best since May — while Brexit odds have drifted to 7/2, equating to a 22% chance.
Betfair spokeswoman Naomi Totten said: “With just three days left until the vote the Betfair market momentum is now all behind Remain, which was backed as low as 1/5 this morning, with one customer backing it to the tune of £100k.