The star has already won an Oscar for her portrayal of Britain’s Queen Elizabeth II in a film set in the aftermath of the death of Diana, Princess of Wales.
She resumed the role for a play which imagines the private weekly meetings between Elizabeth and Britain’s prime ministers over her six-decade reign.
In the production, which has already had a successful run in London, she morphs from a young princess to a modern-day monarch.
Mirren beat fellow British actresses Carey Mulligan and Ruth Wilson, as well as Elisabeth Moss and Geneva Carr for the Tony for best performance by a leading actress in a play.
After her name was called by presenter Bradley Cooper she kissed her husband, director Taylor Hackford.
Arriving at the microphone on stage at Radio City Music Hall in New York, she proclaimed: “Your Majesty, you did it again.”
She then dedicated her award to Hackford, saying: “What a massive, massive honour. Baby, this is for you and you know why.”
Addressing the audience, she added: “It’s nothing rude, incidentally.”
Meanwhile, British actor Alex Sharp was an unknown before he landed one of the most coveted Tony awards over the likes of Hollywood stars Bradley Cooper and Bill Nighy.
The 26-year-old won the title for lead actor in a play for his starring role as a gifted mathematician with Asperger’s syndrome in The Curious Incident Of The Dog In The Night-Time.
The surprised London-born actor described his triumph, for his first professional acting job, as “insane”. Sharp, who only graduated from Juilliard School for performing arts in New York last year, admitted that he had a “blank resume” before his Night-Time role.
Richard McCabe, who plays prime minister Harold Wilson in The Audience, was awarded best performance by an actor in a featured role in a play and charmed the crowd when he proclaimed “oh my giddy aunt” as he collected his gong. He added: “Were he alive today, I know Harold Wilson would be very tickled.”