The toddler fell from the second floor to the ground floor of the popular shop in Liverpool city centre shortly before 1.30pm today.
She was taken to the Royal Liverpool Hospital where her injuries are being assessed.
Merseyside Police sealed off the store for forensic examinations and described the girl's condition as critical.
A spokeswoman said: "Officers were called to the Primark clothing store in Liverpool city centre following reports that a three-year-old girl had fallen over the side of an escalator and through the gap between the two escalators.
"It appears she may have fallen approximately 100 feet from the escalator going down from the second to the ground floor.
"She was taken to the Royal Liverpool Hospital where her injuries are being assessed. Her condition is critical.
"Police are looking as CCTV possibilities and speaking to witnesses."
The shutters of the store have since been pulled down and police officers have been stationed at the doors.
A spokesman for Primark said: "We are obviously profoundly shocked and saddened to learn of the accident in our Liverpool store this afternoon.
"We are doing all we can to assist the authorities in their investigation.
"All our thoughts are with the little girl and her family."
The store, which at 84,000 sq ft is one of the biggest Primark stores in the UK, opened in April.