Abstract: In order to improve the precision and efficiency of micromanipulation, a physical coordinate system is established automatically by two different circles printed on the physical marker. The circles are recognized by Hough circle algorithm and artificial auxiliary. Then, by combining with the physical coordinate, automatic platform system and the coordinate conversion algorithm developed by ourselves, a method is proposed to automatically realize the detection of the CCD (charge coupled device) deflection angle, the scanning of microscopic image and the repeat positioning of micromanipulation under a microscope. As the experimental results show, the error of two successive positioning can be controlled in a range of 320 pix×240 pix in the center of CCD screen (640 pix×480 pix), that is a quarter of the whole screen, and the success rate can be up to 90%. The repeat positioning of the physical object will be realized precisely under the microscope, while it is only needed to affix the transparent label with two circles to the bottom of the material object, and install the relevant software and hardware.