A method for real-time, quick calibration of four-quadrant photodetector (QD) signals in optical tweezers system is provided. The experimental results indicate that the method is feasible and precise. Its instability is less than 1%. In addition, the effect of the bead depth on calibration factor is investigated experimentally, and it is found that the calibration factor decreases when the bead depth increases. The method can be used to monitor the stability of the optical tweezers system.