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Aiming at the application requirements of fast and high-precision registration of pathological microscopic images, an improved sub-pixel fast registration method is proposed. By using the logarithmic difference function to evaluate the overlap region of the microscopic image, the coarse positioning and effective subgraph of the two images are obtained by H-type logarithmic difference template registration. Then local up-sampling phase correlation is used on the two subgraphs to obtain sub-pixel level registration. The method takes the advantages of fast registration speed of H-type logarithmic difference template to avoid the defect that the calculation of phase correlation method increases sharply with the increase of image size. Besides, the method avoid mismatch caused by the blank image. Experiments show that the improved microscopic image registration method has a registration accuracy of 0.01 pixels and a speed of 3.7 times that of the phase correlation method (image size is 2448×2048), which is more suitable for fast and high-precision matching of pathological microscopic images.