Abstract: The micro-operation robot that works under biological microscope is used for operation of biological cells. Normal position feedback control methods can not be realized in the system because of some reasons. The micro-images accepted by vidicon are the feedback informations for motion that only can be used in the system, and Z-directional informations can not be obtained from the plan images. A method based on the analyzing of frequency characteristics of series micro-images is discussed in this paper, we can get Z-directional informations of the end of micro-tools and operated objects with the method, the calibration and position control can also be implemented with it.