Abstract: A novel method to measure duty cycles of rectangular gratings and evaluate the spatial uniformity of duty cycles was presented on the basis of top-down scanning electron microscope(SEM) images. This method first conducts image grayscale profile preprocessing on target images, then performs border detection based on dynamic programming and least-squares spline approximation, and finally computes the mean and standard deviations of duty cycles in the target area in light of the provided evaluation standards. Using this method, we can not only quickly and accurately measure grating duty cycles and perform quantitative analysis on the spatial uniformity of duty cycles, but also avoid damaging sample preparation process, which greatly make up the deficiency of traditional method. A top-down SEM image processing software, GradUI, for detecting duty cycles of rectangular gratings was developed. The top-down SEM images of several 1 200 lines/mm self-made gratings were processed with the software, and the root-mean-square deviation of the average duty cycle and the estimated value was 0.017 3. The results show that the approach to calculate duty cycles based on top-down SEM images is feasible and of high efficiency, and the software is robust.