The stiffness of optical trap is an important parameter to describe the mechanical properties of the optical tweezers,whose measurement accuracy is almost determined by the calibration accuracy of trap stiffness.A calibration method of optical trap stiffness based on Hough transform is proposed.Firstly,the basic principle to solve the displacement and calibrate the stiffness based on the mean square displacement method by using the Hough transform was introduced.Then,the optical tweezers system was built,and a feasibility analysis was carried out.The measured results are compared with those using the traditional CCD correlation algorithm.When the optical magnification is 120,and the average value of the stiffness measured by the Hough transform method is,and that using CCD correlation algorithm is about .The results are almost the same.The research results show that the Hough method is stable and reliable,and can be used to calibrate the optical trap stiffness.