We instrumented a combined fluorescence spectroscopy and imaging system to characterize the single- and two-photon excited autofluorescence in epithelial tissue. Single-photon fluorescence (SPF) are compared with two-photon fluorescence (TPF) measured at the same location in epithelial tissue. It was found that the SPF and TPF signals excited at corresponding wavelengths are similar in nonkeratinized epithelium, but the SPF and TPF spectra in the keratinized epithelium and the stromal layer are significant different. Specifically, the comparison of SPF signals with TPF signals in keratinized epithelial and stromal layers shows that TPF spectral peaks always have about 15-nm redshift with respect to SPF signals, and the TPF spectra are broader than SPF spectra. The results were generally consistent with the SPF and TPF measurements of pure nicotinamide adenine dinucleotide, flavin adenine dinucleotide, keratin and collagen, the major fluorophores in epithelium and stroma, respectively. The double peak structure of TPF spectra measured from keratinized layer suggests that there may be an unknown fluorophore responsible for the spectral peak in the long wavelength region. Furthermore, the TPF signals excited in a wide range of wavelengths provide accurate information on epithelial structure, which is an important advantage of TPF over SPF spectroscopy in the application for the diagnosis of tissue pathology.