A new method is proposed for determination of three-dimensional orientation of the transition dipoles of single molecules. Water layer is added upon the immersion mirror objective on the total internal reflection fluorescence microscopy. The different indices of refraction between water and oil can lead to optical path difference resulting in defocused imaging. A polarization-displacement prism was added before CCD. Beam was split according to the polarization-displacement prism. The field distribution on the CCD includes the information about the orientation of single molecules. Exact field distribution excited by evanescent waves in CCD was considered. The three-dimensional orientation of single molecule was obtained by analyzing the different images of the orthogonal polarization of light.