We studied the time-resolved polarization-dependent fluorescence spectroscopy of receptor-targeted contrast agents (Cybesin and Cytate) bound with prostate cancer cells in prostate tissue. An analytical model dealing with highly viscous tissue media was developed and used to investigate the rotation times andfluorescence anisotropies of the receptor-targeted contrast agents in prostate tissue. The differences of rotation times and fluorescence anisotropies were observed for Cybesin (Cytate) in cancerous and normal prostate tissues, which reflect changes of the microstructures of cancerous and normal tissues and their different bound affinity with contrast agents. The preferential uptake of Cytate (Cybesin) in cancerous tissue was used to image and distinguish cancerous tissue areas from normal tissue areas. The fluorescence polarization difference imaging technique was used to enhance the image contrast between the cancerous and normal tissue areas. This research may help to introduce a new optical approach and criteria for prostate cancer detection.