The left-back was assessed by the national team's medical staff on Saturday but they felt he had not recovered sufficiently from the ankle problem which forced him to miss Friday`s opening qualifier away to Moldova.
The news on Cole's clubmate John Terry was less definite, though, with the Football Association confirming he would continue to receive treatment on an ankle injury sustained in the 5-0 win over Moldova in Chisinau.
An FA statement released this afternoon read: "John Terry is continuing to receive treatment from the England medical team, having hurt his ankle in the World Cup qualifier with Moldova on Friday.
"Ashley Cole was assessed by the medical staff on Saturday and will not be available for the qualifier with Ukraine on Tuesday."
Cole trained with Chelsea on Friday but a decision has been taken not to risk him against Ukraine, who England played in their final group game at Euro 2012 in the summer.
England finished the game against Moldova with 10 men after Terry limped out of the action in the closing stages,
If Terry is unable to prove his fitness, Gary Cahill is likely to replace him and partner Manchester City's Joleon Lescott.
It would be Cahill's first appearance at Wembley since he suffered the broken jaw against Belgium which ruled him out of the European Championship.
Phil Jagielka, who partnered Lescott when team-mates at Everton, is the other player in contention should Terry be sidelined.