A blood flow-mediated skin fluorescence spectrum (FMSF) detection system was developed to noninvasively detect the dynamic changes of the fluorescence spectrum of nicotinamide adenine dinucleotide (NADH) in the human skin. The fluorescence probe in the system was simulated and designed to improve the collection efficiency of the NADH fluorescence. First, we constructed a seven-layer human skin structure model and used the Monte-Carlo method for simulating the transmission of photons in tissues. Then, we investigated the influence of the core diameter of the collection fiber-optics, distance between the excitation and collection fiber-optics, and distance between the fiber-optics probe and skin on the fluorescence spectrum detection. Finally, we developed a fiber-optics probe according to the simulation results and applied it to the skin NADH fluorescence spectrum detection. The results show that the NADH fluorescence spectrum can be measured using the FMSF detection system with post occlusion reactive hyperemia, and the NADH fluorescence intensity shows a dynamic change. Compared with the existing technology, the skin NADH detection method based on FMSF technology has the advantages of rapid measurement and noninvasion; thus, providing advanced technology for studying mitochondrial energy metabolism, diabetes, and other related diseases.