Fecal contaminations on poultry carcasses, not easily discemable by human eyes, are potential harbor sites of pathogenic Escherichia Coli (E. coli O157:H7). Development of sensitive detection methods for fecal contamination is essential to ensure safe production of poultry products. Fluorescence has been shown to be very sensitive in detecting fecal and other biological substances that can harbor pathogens. In this study, fluorescence excitation-emission spectra of poultry fecal matter were compared with spectra for poultry skin and meat. Results indicated that the combinations of fluorescence intensities at the wavelength of 520 nm, 579 nm, 625 nm, and 635 nm with 411 nm excitation showed above 97% accuracy for differentiation of the contaminants from poultry tissues. Excitation and emission bands determined could be used for constructing a real-time fluorescence imaging device for detection of harmful residues on poultry carcasses.