A new method is developed for two-photon fluorescence anisotropy imaging. Its biological applications are tested. This system is based on a two-photon laser scanning fluorescence microscope. A polarization beam splitter was inserted into the optical path to separated fluorescence into components of orthogonal polarization. By rotating the polarization of the excitation beam by 90 ° , four images were collected for each sample. These images were then processed pixel-by-pixel to generate a new fluorescence anisotropy image. The capability of this method is tested for different samples, including FITC, FITC-CD44 in solution and FITC-CD44 attached to the membranes of tumor cells. The results show that fluorescence images can distinguish fluorescence molecules of different molecular mass, or detect changes in microenvironment.