The present invention discloses a positioning method of a minimally invasive osseous surgery guiding channel. The method comprises the steps of: (1) arranging a reference point and a reference scale which can be displayed in an X-ray photograph, and taking the X-ray photographs of an area for surgery including the reference point and the reference scale in two dimensions; (2) determining pixel point coordinates of two guiding points in each X-ray photograph according to X-ray photograph data including the reference point and the reference scale in the two photographs; and (3) computing three-dimensional coordinates of the two guiding points according to the taken X-ray photograph data, the reference point, the reference scale and guiding point data, and determining the position and direction of a straight line, that is the guiding channel, connected between the two guiding points. According to the method, the position and direction of the minimally invasive osseous surgery guiding channel can be rapidly determined, a patient only needs to take X-ray photographs for two times, continuous multi-angle X-ray photographs or three-dimensional CT reconstruction via whole-body scanning for determining the position and direction of the guiding channel are not required, and therefore radiation of X-ray or CT scanning on doctors or patients is reduced. The method is simple and practicable.