An audience of 20 million people watched Usain Bolt win gold in the men's 100m final, according to BBC figures.
Overnight ratings showed 19.4 million tuned in to BBC1 to see the race, with another 600,000 watching via the red button service.
The BBC's Olympics boss, Roger Mosey, said the figures were "sensational".
Last night's audience was the biggest for the games so far, outstripping the 17.1 million who watched Mo Farah win the 10,000m on Saturday night.
The games have consistently pulled in massive audiences for the BBC, with more than 16 million people watching Jessica Ennis triumph in the heptathlon and 15.6 million watching Greg Rutherford's long jump gold.
Figures released by the corporation also show record numbers watching the coverage online with its website recording a peak audience of 8 million - above the previous record of 5.7 million.
More than 1.5 million people have downloaded the BBC's Olympics app for their phone and around 17 million people have used one of the 24 red button streams.
The website has seen 29 million requests for its interactive video streams with video of Bradley Wiggins' win being the single most popular, with 729,000 requests.
Mr Mosey told followers on Twitter that Andy Murray's victory over Roger Federer in the men's singles was watched by 10.7 million people.