Ambient temperature variation is one of the key factors that affect the measurement accuracy of Shack-Hartmann wavefront sensor (SHWS). The influence of the distortion of micro-lens array (MLA) and the refractive index variation, the distance changes between MLA and charge coupled device(CCD), and the distance change between spherical point source and SHWS on measurement accuracy of SHWS are analyzed by using the thermal analysis function of Zemax software, which is caused by ambient temperature variation. The analysis and calculation show that the main factor affecting the measurement accuracy of SHWS is the distance change between MLA and CCD caused by ambient temperature variation. The measurement error is 0.52 nm root mean square(RMS) when the ambient temperature rises every 1 ℃ from 21 ℃ to 24 ℃. The experiments of testing the measurement accuracy of SHWS are carried out by using a nearly perfect spherical wave generated by single mode optical fiber diffraction. The results obtained from simulation and experiment are basically identical.