The present invention relates to a non-invasive positioning system for screwing and fixing a bone, where a intramedullary nail is inserted to marrow of the bone, and the intramedullary nail comprises a wall and at least one through-hole through the wall for screwing and fixing by at least one corresponding set screw, the system comprises: an in vitro locator having at least one light source to emit a laser with a wavelength to the muscle tissue to form an incident light and running through the muscle tissue and the bone to form a penetrated light, an optical holder having an optical lens and a positioning ring portion for removably disposing the optical lens, wherein the focusing spot of the incident light, the focusing spot of the penetrated light, and the at least one through-hole are aligned in a line to confirm a linear position for screwing and fixing the intramedullary nail.