More than 31,000 people voted in the poll for the organization's "British Hit Singles" book.
The six-minute epic first topped the charts in 1975 and became No. 1 again in 1991 when a fund-raising version was released after the death of the group's singer Freddie Mercury.
The award was accepted Wednesday by Queen drummer Roger Taylor and guitarist and songwriter Brian May at London's Dominion Theatre.
"The song was apparently so far ahead of any rival that even months before the deadline, its position in this new poll was unimpeachable. Thank you, people, for the memory," said May.
The song, written by Mercury, also continues to live on every night at the musical "We Will Rock You" which is running at a London West End theater.
In all, the song has spent a total of 14 weeks at the top of the UK charts. Because of its complex harmonies it was difficult to reproduce and the band were unable to perform it live.
No. 2 in the top 100 list was John Lennon's "Imagine" which joined four other Beatles' hits in the top 10.
The most recent single to feature in the chart was Robbie Williams' "Angels" which was released in 1998, while the Spice Girls' "Wannabe" was voted eighth most popular.
The top 10 favorite singles of all time:
1. Bohemian Rhapsody - Queen
2. Imagine - John Lennon
3. Hey Jude - The Beatles
4. Dancing Queen - Abba
5. Like a Prayer - Madonna
6. Angels - Robbie Williams
7. Penny Lane/Strawberry Fields Forever - The Beatles
8. Wannabe - Spice Girls
9. Yesterday - The Beatles
10. Let It Be - The Beatles