In order to improve the precise positioning of micro parts, focusing-Zernike matrix algorithm were proposed. Firstly, coarse and fine combination focus function was established based on the Krisch edge detection operator and Sobel method. Secondly, focusing window was selected dynamically, and the factor was corrected target size of clarity evaluation function, focus direction was judged by improved hill-climbing algorithm. Thirdly, improved Zernike moment was determined the maximum circumscribed unit circle based on the normalization of the target point, and the pixel was judged edge point using edge parameters, then the process was given. Experimental simulation was done by the automatic alignment and stacked microassembly task of two micro cylindrical cavity to analyze the algorithmic. The results showed that the dynamic adjustment algorithm had a clear image and good alignment, and the maximum value of absolute error in x and y directions were 0.032 5 and 0.032 1 pixels per inch, respectively, which had higher positioning accuracy than other algorithms.