Abstract: Aiming auto-focusing system, auto-focus algorithm based on cross correlation was brought forward. The limitation of conventional auto-focusing in definition and velocity was analyzed. Correlation principle was introduced, and template correlation algorithm based on cross correlation was defined. Owing to difference of template image selected, the result of experimentation is different, so that auto-focus analysis algorithm is different. Based on different template image and the result of experimentation, two forms of correlation function were presented, including the first template correlation and the second template correlation. If the place of template image is before focusing, the form is named the first form, curve of which changes from small value to big, then to small, if the template image is focusing image, the form is named the second form, curve of which changes from big value to least, then to big. The result which two forms processes image was compared with the result of experimentation of other focus evaluating functions, such as square gradient function, correlation function. By experimentation comparison, it concludes that the auto-focusing algorithm based on cross correlation improves definition and velocity of auto-focusalgorithm. The algorithm has been successfully tested to be effective on micro-fluidic alignment assembly auto-focusing system. It shows that the algorithm has achieved better auto-focus effects.