The larger the magnification of the microscope, the smaller the field of view. It is usually necessary to obtain a slice image with a large field of vision and high resolution through image stitching. Microscopic images are characterized by rich features, a large amount of information, and contain several similar regions, resulting in high time consumption and low registration accuracy. This study proposes a mosaic algorithm for microscopic images, which can quickly perform registration and fusion. In this study, template matching is used for initial motion estimates and the improved optical flow approach is employed for further matching. After collecting the matching point pairs, the mismatched points are removed using similarity and position restrictions, which increases registration accuracy. Experimental results show that the proposed method can effectively improve registration accuracy while maintaining real-time performance.