The invention discloses a three-dimensional box positioning system and positioning method for an automatic robot welding assembly line. The system comprises a conveying device, a jacking and positioning device, a front retaining and positioning device, a back pushing, clamping and positioning device and a synchronous clamping and centering device. The front retaining and positioning device, the back pushing, clamping and positioning device and the synchronous clamping and centering device are arranged at the upper end of the jacking and positioning device. Three-dimensional accurate positioning of welded boxes on the automatic assembly line can be realized effectively, high automation degree and high controllability are realized, and stability and reliability in a robot welding process are improved.