The Football Association announced on Wednesday that the Liverpool full-back is set to return to Merseyside to recover from a back injury.
Alexander-Arnold has been dealing with the problem for a couple of weeks and it has been decided that would have likely kept him out of upcoming matches with Czech Republic and Montenegro.
"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 UEFA Euro 2020 qualifiers," the FA statement read.
"The 20-year-old will now continue his rehabilitation at his club leaving Gareth Southgate with a 22-man squad."
— England (@England) March 20, 2019
Alexander-Arnold received his first call up ahead of the World Cup last May after breaking through in the Reds' run to the Champions League final.
The defender scored his first international goal against the U.S. national team in November, becoming the youngest England goalscorer since Michael Owen in the process.
This season, the defender has made 29 total appearances for Liverpool in all competitions as the Reds sit atop the Premier League and in the quarter-finals of the Champions League.
With Alexander-Arnold's absence, Gareth Southgate could look to Kyle Walker or Kieran Trippier to play at right-back.
He is the fifth player to withdraw from the squad since it was announced, with Luke Shaw, John Stones, Fabian Delph and Ruben Loftus-Cheek all set to miss out.
Southampton midfielder James Ward-Prowse and Chelsea teenager Callum Hudson-Odoi have been added to Southgate's squad as cover, but the temptation to call-up Crystal Palace's Aaaron Wan-Bissaka has been resisted despite Alexander-Arnold's withdrawl.
England are set to face Czech Republic at Wembley Stadium on Friday before travelling to Montenegro on Monday as they look to follow-up an encouraging 2018.