A new application of a fluorescent sensor (PyDPA) for the discrimination of redox-responsible molecules is reported. Nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD(+)/NADP(+)) and flavin mononucleotide/flavin adenine dinucleotide (FMN/FAD) were differentiated by means of ratiometric fluorescence change from excimer-monomer equilibrium and time-dependent fluorescence change, respectively.