An optical calibration system including a calibration module, a light source, an optical sensing device and a calculation module is provided. The calibration module includes a calibration plane and a plurality of calibration structures between the calibration plane and the light source, wherein the calibration structures are disposed at predetermined calibration positions. The light source projects a linear light section toward the calibration module. The optical sensing device senses reflected light from the calibration plane and the calibration structures reflecting the linear light section to generate a sensed frame. The calculation module calculates positions of gravity centers of a plurality of calibration points in the sensed frame corresponding to the calibration structures.