The misalignment aberration in the hig h-precision testing of spherical surface may introduce a significant error in t he measurement results,especially for the test surfaces with high numerical aper ture,and they are difficult to be completely removed from the meaasured data wit h the traditional misalignment calibration method.This paper presents a high-pr ecision way based on the difference method for the calibration of spherical surf ace misalignment,in which significant high-order aberrations introduced by misa lignment can be observed.With the different wavefront obtained from the measured wavefronts corresponding to two slightly different defocusing amounts,the high -order aberrations due to misalignment are obtained and separated from the wave front under test,by which high-precision misalignment calibration can be realiz ed.The experiment with Zygo interferometer is carried out to demonstrate the fea sibility of the proposed method.Both the wavefornt defocus and tilt are introduc ed in the high-numerical-aperture spherical surface under test,and an accuracy of root-mean square value of 0.0015λ and peak -to-valley value of 0.0189λ is ach ieved with the proposed misalignment calibration method in the experimental vali dation.The proposed method requires no foreknowledge about the testing system an d testing surface,lowers the requirement on the test surface adjustment,and it i s of significant applicability for the high-precision shperical surface testing .