16-bit images are commonly used in some image processing and analysis with high precision, we need to adjust the window to obtain the information of interest for the large gray range. Manually adjusting the window requires users’ rich experience to get a clear image, and will take some time, so it will reduce the efficiency of users. The existing auto-window algorithms cannot process the images of various histogram distributions very well. In view of this situation, an auto-window algorithm for 16-bit grayscale image based on the analysis of a large number of 16-bit images is proposed. After reading the image, the algorithm identifies the type of the image’s histogram automatically and calculates the best window width and level. A lot of experiments are performed to the proposed algorithm and compared with the existing window algorithms, the result shows that the method can significantly improve the images’ clarity and contrast and has better performance than the conventional window method.