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The comedian, author and TV personality appeared on stage at the BBC show, titled Platinum Party at the Palace, to introduce Prince Charles and Camilla, Duchess of Cornwall to the stage.
Beforehand, he delivered a speech in which he praised the Queen for “tolerating prime ministers” throughout her 70 years on the throne.
“As a nation, we have been lucky, lucky, lucky above all others to have had our Queen to represent us over 70 years of Trooping the Colour, royal tours, Christmas Day at three in the afternoon,” Fry said.
He continued: “How many local sewage works has our Majesty opened with a bright smile? How many plaques unveiled? How many trees planted, ribbons cut, ships launched? How many prime ministers tolerated – for that alone, no admiration is high enough.”
His last comment about “prime ministers” drew gasps as well as laughter from the crowd.
Elsewhere during the ceremony, comedian Lee Mack ad-libbed a joke about Boris Johnson and the Partygate scandal while introducing DJ Jax Jones – in front of the prime minister himself.
Later in the ceremony, viewers noticed that George Ezra censored his own song lyric about death during a performance of one of his hit songs.
The ceremony was kicked off in heartwarming fashion as the Queen showed off her acting skills for a “cute” skit opposite the CGI version of Paddington Bear voiced by Ben Whishaw.
Catch up with all the live updates from the event as they happened here.