Each chicken carcass intended for U.S. consumers is mandated to be inspected by Food Safety and Inspection Service (FSIS) inspectors for its wholesomeness at the processing plants. Fluorescence responses of wholesome and unwholesome chicken carcasses were characterized and further evaluated for potential on-line applications for detection and classification of wholesome and unwholesome chicken carcasses. For this study, unwholesome chicken carcasses included cadaver and those with disease conditions such as airsacculitis and septicemia. Fluorescence characteristics from the epidermal layers in the breast areas of chicken carcasses were dynamic in nature. Emission peaks and ridges (maxima) were observed at 386, 444, 472, 512, and 554 nm and valleys (minima) were observed at 410, 460, 484, and 538 nm. One of the major factors affecting the line shapes of fluorescence responses from chicken carcass skin layers was absorption by hemoglobin. With the use of the normalized ratio spectra (NRS) approach, oxyhemoglobin was shown to be a major constituent in chicken carcasses affecting the fluorescence emission line shapes. Subtle line shape changes in the NRS also provided a qualitative means by which to assess the minute differences in oxy- and deoxyhemoglobin compositions perturbed by poultry diseases such as septicemia and airsacculitis. With the use of simple fluorescence band ratios as a multivariate model, wholesome and unwholesome chicken carcasses were correctly classified with 97.1% and 94.8% accuracies, respectively. On-line implementation of fluorescence techniques for the assessment of chicken carcass wholesomeness appears promising.