An auto focusing method based on auto focusing window selection was presented according to the imaging principle and characteristics of fundus images.The preliminary focusing window is extracted through the focusing window selection algorithm based on local entropy with improved threshold.The final window is located through multi-directional stepping algorithm.The image sharpness evaluation function combined with the characteristics of local contrast sharpness evaluation function and gradient vector square function was presented according to low contrast fundus images.Experimental results show that the presented auto focusing window selection algorithm ensure the stability and accuracy for the focusing window,and the image sharpness evaluation function has better sensitiveness for low contrast fundus images.