A biosensor model was developed for a commercial NADH fluorescence probe to describe the single-frequency excitation and emission fluorescence behavior of an aqueous mixture of fluorophores. This model is essential in correlating the measured signals to the concentrations of fluorescent compounds in a bioreactor. In addition to the concentrations of fluorescent components, the relevant parameters of the model are the absorbance at both the excitation and the emission frequencies by the solvent and other absorbing species, the background signals, the light path length of the bioreactor vessel, the fluorescence yield, and the lampdetector configuration. Due to inner-filter effects and other interferences, the probe signal is intrinsically nonlinear in both the fluorophore concentration and the path length. An important parameter in the model is the geometric constant, s, which accounts for variations in the monitoring efficiency throughout the sample because fluorescent light is emitted in all directions. Previous models, derived from an unrealistic assumption that fluorescent light is emitted only in one direction parallel to the probe axis, are shown to be seriously deficient. The validity of the model was verified experimentally for a single-component solution in which both the fluorophore concentration and path length were varied.