Autofluorescence of rabbit and human epithelial tissues were studied by using a depth-resolved fluorescence spectroscopy system with multiple excitations. Keratinization was found to be common in the squamous epithelium. Strong keratin fluorescence with excitation and emission characteristics similar to collagen were observed in the topmost layer of the keratinized squamous epithelium. The keratin signal created interference in the assessment of the endogenous fluorescence signals (NADH/FAD fluorescence in epithelium and collagen fluorescence in stroma) associated with the development of epithelial precancer. Furthermore, the keratinized epithelial layer attenuated the excitation light and reduced the fluorescence signals from underlying tissue layers. The autofluorescence of columnar epithelium was found to be dominated by NADH and FAD signals, identical to the autofluorescence measured from nonkeratinized squamous epithelium. The study also demonstrated that a fluorescence signal excited at produced sufficient contrast to resolve the layered structure of epithelial tissue, while the signal excited at provided the information for a good estimation of epithelial redox ratios that are directly related to tissue metabolism. Overall, the depth-resolved measurements are crucial to isolate the fluorescence signals from different sublayers of the epithelial tissue and provide more accurate information for the tissue diagnosis.