The macroscopic calibration method cannot be applied in the microscopic three-dimensional digital image correlation system due to the small depth of field and the complex optical paths of a stereo microscope. As for this problem, a fixed-point rotation calibration method is proposed, which is suitable for microscopic stereo vision. The mathematical model is established based on the relationship between magnification and depth of field, and thus the maximum angle between calibration plate and XY plane is obtained. In addition, the fixed-point rotation platform is designed to calibrate the calibration plate. The calibration parameters are optimized by a series of experiments and the inclination angle used for minimizing the overall error of the calibration parameters is obtained. The calibration results show that the main point coordinate error is not larger than 1.8 pixel, the maximum deviation of the relative translation vector of Z component is less than 0.15 mm, and the calibration result tends to be stable when the attitude number is 10 or above. The accuracy of the calibration results is tested by use of a precision displacement platform and the results show that the average relative error of displacement measurement by the proposed method is 2.2% and the mean square error is 0.36 μm.