Enzymatic turnovers of single cholesterol oxidase molecules were observed in real time by monitoring the emission from the enzyme＇s fluorescent active site, flavin adenine dinucleotide (FAD), Statistical analyses of single-molecule trajectories revealed a significant and slow fluctuation in the rate of cholesterol oxidation by FAD. The static disorder and dynamic disorder of reaction rates, which are essentially indistinguishable in ensemble-averaged experiments, were determined separately by the real-time single-molecule approach. A molecular memory phenomenon, in which an enzymatic turnover was not independent of its previous turnovers because of a slow fluctuation of protein conformation, was evidenced by spontaneous spectral fluctuation of FAD.