Fluorescence technique of evanescent field has been widely used in the research work of industry, biology and medicine. In this paper, rigorous electromagnetic theory of evanescent field distribution in various layered structure is dealt with. By means of the law that stimulanted fluovescence intensity is proportional to the energy of local electrical field and the measured relative fluorescence intensity, the distance between cell and glass substrate can be derived. Some applications of fluorescence measurement of evanescent field are also presented in this paper.