Abstract: This paper proposes a three-dimensional scanning probe calibration method to reduce the positioning accuracy requirements.A fast least squares iterative algorithm and an iterative data filtering algorithm are designed to achieve the highly precise and efficient calibration of the three-dimensional scanning probe.Several calibration simulation experiments are designed to verify its stability and accuracy,and the influences on calibration result from various errors are tested.Experimental results show that this three-dimensional scanning probe calibration method does not require the positioning accuracy of measuring machine,it is insensitive to the measuring machine error and scanning probe detection error,the accuracy of unit vector of coordinate system of three-dimentional scanning probe reaches 0.000 01,and the three-dimensional scanning probe calibration requirements are completely satisfied.