The tightness was in the same hamstring he injured earlier in the season, which kept him out of action at the turn of the year.
Gunners boss Arsene Wenger said: "We cannot even take a gamble, so he is not available."
Veteran defender Sol Campbell did, however, join in training and he looks set to recover from a groin problem in time to face the Portuguese side, who will take a 2-1 lead to the Emirates Stadium for the second leg of the last-16 tie.
Cameroon international Alex Song is available as his two-match suspension does not apply in Europe.
Andrey Arshavin and Abou Diaby could both feature after coming off the bench against Burnley, while Mexican Carlos Vela could return to the squad after international duty.
William Gallas (calf) is still sidelined, while Aaron Ramsey (broken leg), Robin van Persie (ankle) and Johan Djourou (knee) are long-term absentees.
Gunners (from): Almunia, Clichy, Vermaelen, Campbell, Eboue, Denilson, Nasri, Song, Walcott, Arshavin, Bendtner, Fabianski, Sagna, Diaby, Eduardo, Traore, Eastmond, Silvestre, Vela.
Arsenal vs Porto To Qualify: Arsenal 4/6