An automatic positioning system using a pair of two-dimensional (2-D) zero-reference gratings was established to implement the nano-positioning in 2D directions. Firstly, the 64×64 coded 2-D zero-reference gratings with a grid width of 5 μm was designed and fabricated. Then, the photoelectric conversion circuit and control circuit were built for the automatic nano-positioning system to implement data acquisition, display and motion control and other functions. Finally, an automatic positioning algorithm using an integral method was proposed to achieve the automatic positioning in 2D directions. The results show that the system positioning speed depends on the transmittance of light intensity distribution and the positioning success rate is related to the starting position. If the starting position is located in the central circular area with a radius of 2.5 μm, this equipment may achieve nanoscale positioning. If the starting position is located in the central circular area with a radius range of 5—200 μm, the automatic alignment may present blind spots. In this case, the starting positions should be changed to perform the positioning process again and the alignment will be achieved successfully.