Liverpool defender Alexander-Arnold was in line to start Euro 2020 qualifiers against Czech Republic and Montenegro.
But he has joined Ruben Loftus-Cheek, Fabian Delph and John Stones in pulling out of the squad due to injury.
"The right-back arrived nursing a back injury and, despite progress in recent days, it was unlikely he would be able to play a role in the forthcoming EURO 2020 qualifiers," an FA statement said.
"The 20-year-old will now continue his rehabilitation at his club leaving Gareth Southgate with a 22-man squad."
With no replacement called up, Kieran Trippier and Kyle Walker will likely compete to line up on the right side of England's defence.
Czech Republic visit Wembley on Friday with Nations League semi-finalists England heading to Montenegro three days later.