The selection of the focusing window is the key procedure in achieving precise automatic focus of the microscope. For the traditional focus window selection method, the limitation is that the target object cannot be accurately positioned. This paper proposes an improved artificial fish focusing window method. The method takes the area-of-interest of the whole image as the basis of the selection window. Through utilizing the global optimization ability of the artificial fish swarm algorithm, the best focusing window can be obtained. Adding the global optimal value to the behavior update of each artificial fish makes the artificial fish quickly move to the optimal position. Under the premise of ensuring accuracy, the elimination behavior is introduced to improve the convergence speed of the algorithm in the later period. Furthermore, according to the characteristics of the bulletin board in the algorithm, the interference area is identified with the trend comparison method, and the influence of the non-target area is effectively excluded. Experiment results show that the focusing window obtained by this algorithm can be well-suited for the target object, greatly improve the accuracy of autofocus, and make the efficiency improvement 1.65 times than the traditional method.