Auto-focusing is one of the key issues in the non-mydriatic fundus camera, a fine and rough combined auto-focusing method is presented after analyzing imaging principle of the fundus camera and the retinal image characteristics. Inscribed square fixed focusing window selection method combined with characteristics of fundus image field is presented, and Robert gradient function is used to realize the rough focusing. The focusing window for fine focusing is selected through auto-focusing window selection algorithm based on local entropy, and image sharpness evaluation function based on Zernike orthogonal moment with 9 pixel×9 pixel mask is presented to realize the fine focusing. The experimental results show that the focusing window method based on inscribed square is more stable, and proposed image sharpness evaluation function for fine focusing has better sensitiveness and realtime performance.