The invention discloses a spine three-dimensional positioning method and relates to the technical field of minimally invasive surgery. The method includes: acquiring a normal position image and a sideposition image of a spine through X-ray photography; guiding in an X-ray photography image, and determining spatial positions of mark points C and D in each of the normal position image and the sideposition image according to a given scale; respectively marking original point and coordinate system in the normal position image and the side position image; marking related points in the images, andcalculating to generate coordinate values of each point by a system; determining a spatial straight line according to two points, calculating spatial coordinates of the related points and included angles between the straight line determined by the points C and D and a horizontal plane and a vertical plane, and determining puncture position, angle and distance of a puncture needle through a triangular function; according to output parameters, setting position and angle of the puncture needle of a spine puncture locator. By the method, complex calculation process is made quite simple and convenient, and visual puncture line prompting is combined, so that errors caused by calculation are avoided basically.