The detection error of Hartmann-Shack (H-S) wavefront sensor is one of the main errors in adaptive optical systems. In this paper, the detection error of H-S wavefront sensor based on the electron multiplying charge-coupled devices (EMCCD) is firstly analyzed and the detection error formula is established. Furthermore, the numerical simulation and experimental results show that the centroid detection error is related to the gain of EMCCD. The centroid detection error decreases with gain increasing when the gain is lower than Pmax/(Q·Nmean). When the gain is higher than Pmax/(Q·Nmean), the centroid detection error increases with the gain increasing, where Pmax is the peak electric charge value of EMCCD, Q is quantum efficiency, Nmean is the mean number of incident photons.