A surgical instrument positioning system and a positioning method thereof are provided. The natural feature information datum a of the human body presented by a medical image (for example, CT) taken before surgery is first input into the positioning system, and 3D datum reconstruction is performed. The position information of the hand-held instrument, obtained by the light sensing element, is then transmitted back to the positioning system. Then, the feature points can be automatically suggested via the positioning system or the feature points can be automatically designated or the feature points can be manually selected by the operator to establish the coordinate relationship between the patient position and the hand-held instrument, thereby solving the problem of the impact of the prior art on the surgical operation.