The threshold selection method for the spot image of Shack-Hartmann Wavefront Sensor (SH WFS) has a large influence on the centroid detection accuracy. This paper analyzes the features of spot image of a SH WFS,and studies the effect of the threshold of spot image on the centroid detection accuracy,and then it presents an adaptive threshold selection method based on gray histogram principles. The experimental results show that the method can effectively and automatically select the threshold of each frame image when signal-to-noise ratio(SNR) of the spot image of SH WFS is more than 4, but it can not get the ideal segmented image when the SNR is less than 4. The SH WFS with the threshold selection method is used in the adaptive optics system to carry out a closed-loop correction experiment. After correction, the wavefront distortion of 10.41λ is corrected to 0.12λ in PV (λ=632.8 nm), and the system resolution reaches the diffraction limit. These results show the method can meet the practical requirements of SH WFSs.