The enzymatic reaction system with a solid−liquid−gas three-phase interface microenvironment allows oxygen to be directly supplied to the oxidase catalytic reaction from the gas phase, effectively improving enzyme kinetics as compared with the conventional two-phase system. For this new system, a mathematical model is developed in this work to describe the enzymatic reaction coupled with interphase mass transfer, by which the influences of three-phase interfacial microenvironment on reaction kinetics can be systematically and quantitatively explored. The numerical simulations reveal that the flux of oxygen transport across the interface between the gas phase and the enzyme layer dominantly determines the H 2 O 2 production rate. The porous substrate possessing larger porosity and smaller pores, when coated with a thick and concentrated enzyme layer, can potentially lead to higher oxygen supply and hence a higher H 2 O 2 production rate. Moreover, regardless of the pore diameter, the H 2 O 2 production rate remains constant after the porosity is greater than 0.8, and if the enzyme concentration is not less than 5 mol m −3 , the H 2 O 2 production rate no longer changes after the thickness of the enzyme layer is greater than 0.5 μm. This work offers a powerful in silico tool for the investigation of the three-phase enzymatic reaction system. The quantitative results and mechanistic findings will lead to optimized design of this promising system.