Fluorescence techniques have demonstrated great potential for detection of the presence of fecal and other biological substances that can harbor pathogens. We used a recently developed laser-induced fluorescence imaging system (LIFIS) to demonstrate the potential use of fluorescence techniques for detection of a range of diluted poultry feces from various sections of the digestive tract, including gizzard, duodenum, small intestine, ceca, and colon, on processing plant equipment. The use of the LIFIS allowed tunable excitation in the visible with selection of emission wavebands for multispectral imaging. Thus, both fluorescence excitation and emission parameters can be optimized (e.g., 415 nm excitation, and 580 and 630 nm emission bands). The results showed that 1:5 and 1:10 diluted feces samples could be detected with 100% detection rates at the 580 and 630 nm emission bands regardless of feces types. Detection rates for 1:50 and 1:100 diluted samples at the 580-nm band were 96.0 and 89.3%, respectively, and those at the 630-nm band were 94.7 and 84.0%, respectively. Even minute amounts of fecal matter on processing plant equipment surfaces, not clearly visible to the human eye, could be detected.