A calibration method of Shack-Hartmann wavefront sensor based on single-mode fiber beam splitter is proposed in this paper. The parallel beam and sensor were precisely aligned through single-mode fiber coupling to make the standard centroids of spot array closer to the physical position of the optical axis of the micro-lenses. The calibration setup and calibrating procedure were introduced. And 11 spherical waves with different radius of curvatures were used to verify the calibration accuracy. The experiment results and errors were compared. The results showed that compared with the conventional calibrating method by using plane wave, the average deviation of reconstructed wavefront peak to valley value with proposed method was reduced from 1.024λ to 0.667λ, and the root mean square value was reduced from 0.136λ to 0.033λ, which demonstrated that the proposed method significantly improves the reconstruction accuracy.