In order to solve the problem of precise positioning of the center of a circular reverse reflection mark in the calibration of the reference scale of industrial photogrammetry system, a positioning scheme of the center of a circular reverse reflection mark based on microscopic vision and image processing was proposed. The composition and principle of the center positioning system are introduced. According to the reflective characteristics of the reflective mark and its image characteristics under the microscope, the image preprocessing, feature extraction and image processing methods of the center positioning of the mark are analyzed. The magnification of the image, the position of the mark image in the field of view, illumination and other factors which may affect the results of the mark center positioning are tested and analyzed. It is of great significance to standardize the calibration process of the reference scale.