A method was proposed to evaluate visual fatigue in autostereoscopic displays by measuring the pupil diameter of viewers.And a pupil diameter measuring device was presented to record the viewers′ pupil diameter when the viewers watch threedimensional videos.The conclusion was made that the degree of visual fatigue increases as the increase of viewing time according to viewers′ subjective evaluation,and experiments shown that the pupil diameter increases as the increase of the degree of visual fatigue.As a result,the pupil diameter can be used as a new measurement index of visual fatigue.The variation of viewers′ pupil diameter is 0.4 mm when viewers′ visual fatigue is medium which can be considered as a critical value of viewing comfort.