Abstract: Based on character analysis of microscope vision system, an alignment strategy based on the micro-assembly experimental platform composed of three microscope vision systems is proposed, which achieves the automatic alignment of the millimeter sized complex micro parts．The image Jacobian matrix based on several microscope vision systems controlling the micro part movement is derived based on microscope vision model．It is proved that the image Jacobian matrix is a constant when controlling position movement in microscope vision guidance．As active movement of the micro part, online self-calibration of the image Jacobian matrix is completed．The incremental PI (proportional-integral) controller is adopted to control the micro part movement quickly and effectively．The experiment verifies the effectiveness of the proposed algorithm.