Abstract: Concerning the auto-focus system of microscope based on image treatment,this paper proposes three new instructing functions: color ratio,image difference and class variance.It proposes the area method to extend the instructing functions which are defined for single point.The extending method has the character of both average method and extreme method.To evaluate the instructing functions some guidelines are proposed,which include responsible object distance range,distinctness of the peak,distance with extreme point,the best object distance and range of function value.The formula for these guidelines are defined and the method to measure them is proposed,and the guidelines of instructing functions mentioned in this paper is tested and compared.