The second reading of the Accession Proclamation was met with applause and cheers of “God save the King”.
Thousands of onlookers had gathered at noon on Saturday in the City of London to witness the proclamation.
The crowd then sang the chorus of the national anthem, singing King in place of Queen.
Those gathered outside the exchange followed the anthem with three cheers for Charles.
It came after the proclamation was first made outside St James’s Palace at 11am on Saturday.
The second reading of the proclamation was performed by Timothy Duke, the Clarenceux King of Arms, in front of the Lord Mayor, Court of Aldermen, High Officers and members of the Common Council.
It formally announced the death of the Queen and proclaimed the succession of the King.
The Coldstream Guards stood to attention as the proclamation was read out.
After the ceremony finished they marched away to the sound of a drumbeat.
Speaking after the reading had ended, Chrissie Ellis, 61, from north London, said it was “extraordinary” to see the Accession Proclamation read aloud.
“I feel very privileged to have seen it,” she said.
Ms Ellis said that singing the national anthem was the standout moment for her, although it was “really strange” to sing “King” instead of “Queen”.
“It’s just getting used to saying it,” she said.
“You’re so used to singing about the Queen.
“That’s what I’ve been brought up with and known all my life. It felt strange, but also momentous and historic.”
Canadian Emily White, 34, described witnessing the moment as “amazing”.
“It was amazing, I got here at 11.59am, so I got here right as it just started,” she said.
“There was an incredible feeling in the crowd. It was such a solemn moment.
“You really felt that a next era was starting, it was exciting.”
Ms White said the public are “still adjusting” to singing God Save The King.
“I think everyone is still adjusting, but still it was again an exciting moment, ready for the next generation to start,” she said.
However, Ms White added that singing the anthem was the standout moment for her.
“I watched the ceremony this morning at St James’s, but then hearing it said this morning, Charles III, it was very moving.”