The Tottenham defender suffered a slight hamstring complaint in the 2-1 win over Belgium on Sunday.
Dier, 26, has not been included in Gareth Southgate’s 23-man squad for Denmark’s visit to Wembley Stadium as England aim to extend their unbeaten campaign in Group A2.
Harry Winks, Michael Keane, Harvey Barnes and Tammy Abraham have also been excluded from the squad list published by UEFA.
Abraham had arrived late to join the England squad after images of him emerged at a party flouting the government’s ‘rule of six’ regulations with fellow England players Ben Chilwell and Jadon Sancho.
Injuries led to Chilwell’s withdrawal from the squad on Tuesday, when Kieran Trippier also left the camp to face a Federation Football hearing for alleged betting violations.
Southgate: Kane is 100% fit
Southgate said on Tuesday that England “were not risking the players” after Sky Sports News reported of a potential club against country between Tottenham and the national team on the availability of Harry Kane, who treats muscle fatigue.
Spurs boss Jose Mourinho has expressed concerns over Kane’s workload ahead of this month’s international break, but Southgate insisted the forward did not suffer from a specific injury and that he was in line to play against Denmark.
“He was experiencing delayed muscle pain, which can often happen to athletes, but there was no injury at any time,” Southgate said.
“Our problem with Harry going into Sunday’s game was being able to play at the highest level. Now with the addition of a few days of training, that’s not even a problem anymore.
“He’s been in great shape since the end of last week, just before the last game. He would have started the last game but didn’t have enough practice time.
“He was obviously doing well during the game, so he’s fit and available. “
Southgate England in tournament mode
Southgate’s three full-backs don’t bring a lot of thrills and spillovers, but it signals a return to the kind of risk-free football that wins international tournaments, writes Sky Sports’ Adam Bate.
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Watch England vs Denmark live in the Sky Sports Main Event starting at 7pm Wednesday; kick off at 7.45 p.m.