The Arsenal midfielder will not be fit to take part in England's game against Uruguay in Sao Paulo, Brazil.
Alex-Oxlade Chamberlain trained away from the rest of the England squad after being declared out for their crucial Group D match against Uruguay.
The Arsenal midfielder picked up the injury in the World Cup warm-up game against Ecuador, and very nearly ruled out for the entire tournament.
However, England boss Roy Hodgson kept the 20-year-old in the squad in the hope that he would recover in time to make some kind of impact.
Oxlade-Chamberlain has been making good progress in his rehabilitation, but Hodgson will not risk him against Uruguay.
In England's first game of the tournament Liverpool's Raheem Sterling - Oxlade-Chamberlain's replacement in the side - put in an outstanding performance in the number 10 role. Wayne Rooney was shifted to the left to make room for Sterling behind the striker, and the 19-year-old shone in the 2-1 loss.
England will be looking for a similar level of performance against Uruguay, who are coming off the back of a surprise 3-1 defeat to Costa Rica.
Both sides will be looking to pick up three points to give themselves a realistic chance of qualifying. A draw for Uruguay would almost certainly not be enough for them, as they still have to face Italy.
For England, a draw would mean they would be relying on Italy to beat Costa Rica, and then the Three Lions would need to comfortably defeat the Ticos in the final group game to scrape through on goal difference.
Hodgson's men played well against Italy but were unable to get a result. Against Uruguay, all that matters is picking up three points to keep the World Cup dream alive.