For autofocus system of the microscope, this paper presents a hybrid search algorithm combining the mountain-climb search strategy with the approximation function strategy. In this algorithm, the mountain-climb search strategy adopts the two-stage algorithm of rough and fine focusing stage. In the rough focusing stage, the gray variance function is used to approach the focusing position quickly. In the fine focusing stage, the Laplacian function is used to locate the focusing position accurately. The algorithm narrows the focus interval by comparing three pictures and the approximation function strategy is used to fit the best focus in this range. This method greatly reduces the number of images required for autofocus and greatly improves the search accuracy. The experimental results indicate that this algorithm can make the search accuracy better than 1 μm.