A non-invasive positioning system (100, 100a, 100b) for screwing and fixing a bone, the system (100, 100a, 100b) including an intramedullary nail for being inserted to marrow of the bone, wherein the intramedullary nail comprises a wall and at least one through-hole running through the wall for screwing and fixing by at least one corresponding set screw, the system (100, 100a, 100b) further comprising: an in vitro locator having at least one light source (111, 1111, 1112), wherein the at least one light source (111, 1111, 1112) is configured to emit a laser beam with a specific wavelength to the muscle tissue surrounding a bone to be fixed to form an initial light beam which is capable of penetrating the muscle tissue,the bone to be fixed and the at least one through-hole thereby defining a penetrated light beam, wherein the system (100, 100a, 100b) is further configured to sense a light spot of the penetrated light beam, characterized in that brightness of the laser is adjustable; and the system (100, 100a, 100b) further comprises a slide rail for placing the at least one light source (111, 1111, 1112) for rotation, wherein the in vitro locator is movably attached to the slide rail to render the in vitro locator rotatable about 180 degrees with respect to the muscle tissue surrounding a bone to be fixed to search for a position in which the initial light beam is capable of passing through through-hole and search for the brightest position after a light spot of the penetrated light beam is found, an optical holder including an optical lens and a positioning ring portion for removably disposing the optical lens, wherein the optical lens removably attached to the positioning ring portion serves to determine a focusing spot of the initial light beam and a focusing spot of the penetrated light beam in order to position the optical holder on the two spots of the initial light beam and the penetrated light beam, wherein, when the focusing spot of the initial light beam and the focusing spot of the penetrated light beam are aligned in a line, the at least one through-hole is aligned in the same line to confirm a linear position for screwing and fixing the intramedullary nail. an in vitro locator having at least one light source (111, 1111, 1112), wherein the at least one light source (111, 1111, 1112) is configured to emit a laser beam with a specific wavelength to the muscle tissue surrounding a bone to be fixed to form an initial light beam which is capable of penetrating the muscle tissue,the bone to be fixed and the at least one through-hole thereby defining a penetrated light beam, wherein the system (100, 100a, 100b) is further configured to sense a light spot of the penetrated light beam, characterized in that brightness of the laser is adjustable; and the system (100, 100a, 100b) further comprises a slide rail for placing the at least one light source (111, 1111, 1112) for rotation, wherein the in vitro locator is movably attached to the slide rail to render the in vitro locator rotatable about 180 degrees with respect to the muscle tissue surrounding a bone to be fixed to search for a position in which the initial light beam is capable of passing through through-hole and search for the brightest position after a light spot of the penetrated light beam is found, an optical holder including an optical lens and a positioning ring portion for removably disposing the optical lens, wherein the optical lens removably attached to the positioning ring portion serves to determine a focusing spot of the initial light beam and a focusing spot of the penetrated light beam in order to position the optical holder on the two spots of the initial light beam and the penetrated light beam, wherein, when the focusing spot of the initial light beam and the focusing spot of the penetrated light beam are aligned in a line, the at least one through-hole is aligned in the same line to confirm a linear position for screwing and fixing the intramedullary nail.