The invention relates to a stitching wall positioning system based on an image sensor and a positioning method. The stitching wall positioning system comprises at least two single-screen processing modules and a stitching and positioning processing module connected with the single screen processing modules, wherein any single-screen processing module is corresponding to a display unit of the stitching wall, and the single screen processing modules respectively collect the brightness values of all pixel points of the corresponding display units, perform the corresponding processing and obtain the number of the pixel points of light spots and the first position coordinate of the center points of the light spots relative to the display units; the stitching and positioning processing module calculates the second position coordinate of the center points of the light spots relative to the stitching wall, and realizes the positioning. Because the positioning of the stitching wall is realizedbased on the image sensor, the positioning performance is not easy to be affected by long-term usage, and because each single-screen processing module performs processing to the brightness value of the corresponding display unit, the processed data size is small, the processing speed is high and the positioning precision is improved.