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This paper has done the research to the micro vision auto focusing evaluation function and focusing control strategy for micro operation. First of all, the characteristics of microscopic images are analyzed and pre-processed. Then the pixel correlation index is introduced and a new grayscale evaluation function is formed by combining with the gradient function to improve the sensitivity and noise immunity of the function. Finally improved the traditional hill-climbing algorithm. In the coarse tuning stage, a large step size is searched and the curve fitting method is used to quickly locate the near actual extreme value point. In the stage of fine tuning, a small step size is used to locate the focal plane accurately. The algorithm avoids the complex threshold setting problem. Compared with the traditional hill-climbing method, the focusing speed is improved to a certain extent, and the success rate of focusing is greatly improved.