A novel method for measuring the non-orthogonality of the interferometer system in a step and scan lithographic tool is presented. First a linear module among the non-orthogonal factor, scaling factor and process related system error has been developed. By real lithographic process the alignment marks are transferred to the wafer, and after development the positions of marks are read out by optic alignment system in the same lithographic tool. The measurement data are used to calculate the non-orthogonal factor and scaling factor of the interferometer by the linear model according to the least square principle. Experimental results show that by this method with the same wafer in different rotation, the measurement repeatability of the non-orthogonal factor is better than 0.01 μrad, and the measurement repeatability of the y to x scaling factor is better than 0.7×10-6. With different wafer in the same condition, the measurement reproducibility of the non-orthogonal factor is better than 0.012 μrad, and the measurement reproducibility of the y to x scaling factor is better than 0.6×10-6.