This study proposes a static/dynamic automatic focusing method of microscopes based on image clarity evaluation to address the autofocus requirements of microscopes for observing dynamic planar targets. First, following the analysis of the focus-defocus image characteristics, the weighted Tenengrad function evaluation method and image sub-block selection focus window mode are established. Second, the focus search model is established using the simulated annealing theory to target the local maximum problem in the search. Finally, to evaluate the dynamic image, the image blur method is used to determine the dynamic no-reference image defocus level. Based on these, a static focusing model, a dynamic defocus detection and real-time focusing model are established. The experimental verification shows that the static focusing model can make the microscope automatically focus quickly and accurately, whereas the dynamic defocus detection and real-time focusing model can make the microscope meet the requirements of defocus detection and real-time focusing under dynamic observation.