In order to improve the accuracy of identification and localization of cell microorganisms by optical micropositioning system, on the one hand, the hand-eye calibration method should be optimized, on the other hand, the accuracy of global image recognition should be improved. Aiming at those, a two-step method for hand-eye calibration of the system was proposed. Firstly, the origin of the system was determined by calibrating the fixed target, and the transformation relationship of the vision module to the origin of the system was obtained. Then, according to the starting point position of each photograph, the number of photoing and the step size of movement, the transformation relationship of the global image to the origin of the system was solved. Finally, in order to further improve the accuracy of the global transformation relationship, an error correction method based on Fourier transform was used to obtain the error of the visual module in movement,then the error was added into the system for compensation. Experimental results show that after error compensation, the micropositioning system has the error mean value in X-axis direction reduced from 10.23 μm to -0.002 μm, the error mean value in Y-axis direction reduced from 6.9 μm to -0.50 μm, and the average positioning accuracy over 99%. The results show that the proposed method can be applied to the optical micropositioning system for high-precision automated capture of cell microorganisms.