An optical tweezers system which can accurately and quickly calibrate the stiffness of optical tweezers in three dimensions was set up combined with quadrant photodiode detector and power spectral density method. The limited Brownian motion of the trapped particle was recorded by a quadrant photodiode detector, and the stiffness of optical tweezers was calibrated by power spectral density method. The relations of the laser power with the stiffness of a 0.97 μm-diameter silica bead and of the 1 μm-diameter PMMA bead were investigated. The results indicate that the stiffness is proportional to the laser power at the range from 50 mW to 120 mW of laser power for a 0.97 μm-diameter silica bead and at the range from 80 mW to 130 mW for a 1 μm-diameter PMMA bead. This system can be used as a precise force measuring tool for microscopic investigation in biology, physics, etc.