In the course of a public appearance to Lichfield Cathedral on the start of the week, the King was heckled by a man who asked him about his brother's links with disgraced financier Epstein.
The man inquired how long the King had been known about his sibling's association with Epstein, and also raised doubts about whether he had asked the police to "cover up" for Andrew.
Despite the protest, there were throngs of fans and applause for the monarch, who was in the region for an event.
Prince Andrew's associations with Jeffrey Epstein have been under intense scrutiny in recent weeks. The prince, who relinquished his royal duties earlier this month, has strongly denied any wrongdoing.
"For how long have you known about Andrew and Epstein? Did you request the authorities to cover up for Andrew?"
The monarch remained silent but continued meeting fans, some of whom chanted "God Save The King". One fan can be overheard asking him for a hug.
A female bystander outside the cathedral was also observed telling the protester to "go away" and to "be quiet".
The scandal over the Duke of York's connections to Jeffrey Epstein has been reignited by misconduct allegations repeated in Ms. Giuffre's posthumously-published memoir.
In the memoir, the accuser claims she had sex with the prince on three separate occasions, including once with Jeffrey Epstein and "several other minors"
Prince Andrew, who reached a financial settlement with Ms Giuffre in 2022, has consistently denied any misconduct. In 2019, he told BBC Newsnight he did not recall meeting Ms Giuffre "ever", and that they "did not have any sort of sexual contact."
But recently, new inquiries have been raised about how he is able to fund his lifestyle despite not being a active member of the royal family.
The revelation that he has only ever paid a token annual rent on his Royal Lodge has intensified the examination over his living arrangements - even though he made significant contributions up front, including for improvements.
The political party said he should be summoned front of members of parliament to give testimony about his lease of his lodge.
But last week, the authorities refused to give MPs time to debate the prince's status or his residence, despite increasing calls for scrutiny.
Buckingham Palace has not issued a statement on whether Prince Andrew might relocate of his home - or where he might reside.
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