Enzyme-catalyzed reactions have the advantages of excellent selectivity, low cost, and mild reaction conditions, but the slow reaction kinetics limit their practical applications. Herein, a microdroplet generator that can continuously and rapidly generate water microdroplets with tunable size was designed and used for the study of an enzyme-catalyzed reaction in microdroplets. Using glucose oxidase as a model and resazurin as a fluorescence probe, the fluorescence intensity of the collected microdroplets sprayed into the gas phase was 35 times higher than that in the bulk system, demonstrating obvious reaction acceleration in the microdroplets. Mechanistic studies demonstrated that local concentration enrichment and enzyme reorientation at the gas-water interfaces play key roles in the acceleration of enzymatic reactions in microdroplets. Further, the potential application of the reaction system in glucose sensing was investigated. Finally, we also studied the reaction acceleration of enzymic catalysis at the oil-water interfaces. Online measurement of the fluorescence signal of microdroplets sprayed into the mineral oil revealed a reaction acceleration factor of 6.2. It was demonstrated that aqueous microdroplets provided a green, efficient, and convenient methodology for enzyme-catalyzed reactions.