The track, by comedians Frank Skinner and David Baddiel with The Lightning Seeds, knocked last week's chart-topper Solo by Clean Bandit and Demi Lovato off the top spot.
The No. 1 comes 22 years after the song was released in 1996 to accompany the European Championships, which were that year held in England.
Three Lions climbed 32 places in the chart, which is separate from the Official Charts Company's weekly chart, after England beat Sweden in their quarter-final game on Saturday afternoon.
The anthemic track, which has been adopted by England fans once again for the current tournament in Russia, entered the chart last week at number 33.
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Marvin Humes, presenter of the Big Top 40, said: "What an incredible day! It's only right that Three Lions is number one on the chart.
"Our boys in Russia are doing us so proud. The nation has come together and hopefully this is only the beginning of the celebrations."
The Official Vodafone Big Top 40, produced by Global, is said to be the only chart that reflects consumption of music in the UK in real time.
On Friday, the Official Charts Company revealed that Three Lions had re-entered its own top 40 for the first time since the 2014 World Cup.
The track is currently at No.24 after a surge in streaming from England fans.