A focusing method by adjusting two slit mark images was proposed to realize accurate focusing of a non-mydriatic fundus camera for an observed retina image in dark. Two deflection-angle prisms were used to project a slit mark on the retina,then light propagated through the slit mark in two different directions based on the bending of deflection-angle prisms. The slit mark image on retina was broken into two images and the two slit mark images would be taken apart when the fundus was out of focus. The separated distances and directions of the two slit mark images were used to decide the degree and direction of the camera focusing. The fundus camera would be in focus when the images of these two slit marks were adjusted to be in a line. Experimental results indicate that the focusing precision of the non-mydriatic fundus camera by judging the two slit mark images has increased by 50 degree (0.5 m-1) as compared with that by judging the dark retina images,which shows this focusing method has advantages in simple adjustment and high precision.