The present invention provides a calibration method of projection effect. The calibration method of projection effect according to the invention comprises the following steps. The first, step (a) is performed to make an optical navigation system move along a predetermined trace relative to an object plane. Then, step (b) is performed to sense the predetermined trace by a sensor to obtain a projection trace. The next, step (c) is performed to calculate a projection effect value according to the included angle between a reflected light and the object plane. Finally, step (d) is performed to calibrate the projection trace with the projection effect value to obtain a calibration trace which is in proportion to the predetermined trace.