Abstract: A new method of estimating the depth information for micro manipulation is proposed. The depth information is obtained by using high-spatial frequency energy parameters of the defocused images. The algorithm consists of two steps:firstly, the depth function is derived using a sequence of calibration images with an interval of 2μm; secondly, in the practical measurement, the depth can be calculated by using this depth equation when acquiring the energy parameter of the micro-manipulator in the certain position. In the testing experiments, the effect of micro glass needle shape on calibration experiments is analyzed and overcome. This method has been successfully applied to both ranging and rapid auto focusing in the actual micro-manipulation robot named NKTY-MR.