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In order to solve the problem of fixed camera out-of-focus in the vision system in the complex environment of industrial production site, an auto-focusing technique for vision camera is presented, which can be used to preprocess ROI regional cutting, Distributed Fourier transform, and other preprocessing by using the image captured by the host computer in real time. Then the image definition is evaluated by the improved definition estimation function of digital image, and the camera driven by motion mechanism automatically searches the focus position and completes the focusing function. The experimental results show that the sensitivity of the evaluation function is improved after Fourier transform filtering. Compared with several typical evaluation functions, the proposed scheme can effectively hold aside the environment interference, the result of auto-focusing is closest to the standard samples, and can be run efficiently under different object spacing.