The invention discloses a method for high-precision measurement of the sphericity of the micro-spherical probe of a nanometer three-coordinate measuring machine. A tungsten probe with large length-to-diameter ratio and a quartz tuning fork are combined. Scanning probe heads with nanometer-level resolution are constructed based on a precision micro stage. During measurement, the two scanning probe heads are driven by the micro stage to scan the large-section contour of a microsphere in the x-axis direction in a differential manner. Radial error generated in the scanning process can be automatically eliminated in a differential manner, and thus, accurate parameters of the large-section contour of the microsphere can be obtained. After the microsphere is rotated a certain angle, the measurement is repeated to acquire multiple sets of parameters of the large-section contour. A three-dimensional spatial contour of the microsphere is constructed through fitting according to feature points obtained. The geometric parameters of the microsphere are calculated based on the three-dimensional spatial contour. Thus, high-precision measurement of the sphericity of the microsphere is realized. The method has the characteristics of small measuring force, wide measuring range, and high measuring precision.