Abstract: The stiffness of optical trap has become an important parameter to describe the manipulation capacity of optical tweezers to particles, which can be affected by laser power. The equipartition theorem and Boltzmann statistic methods were used to calibrate the stiffness of the built optical tweezers, where an image acquisition method was used to measure the particles displacement, and the calibrated results based on these two methods were compared, which showed good agreement. Furthermore, the influences of sample cells laser power on the stiffness were studied. In order to increase the accuracy of stiffness, the amplification factor of light path and temperature should be considered. Seen from the results, the two calibration methods have similar results; the optical trap stiffness increases linearly with the laser power on the sample cell for lower power 1 mW~20 mW, but shows a tendency toward stabilization for higher power 25 mW~60 mW. Whats more, the calibrations accuracy of light path amplification brings a great impact, a 10% relative variation leads to a 23% offset of stiffness, and the temperature has a little influence, a variation of 0.1 ℃ results in 0.034% variation.