The spatial positioning of enzymes and mass transport play crucial roles in the functionality and efficiency of enzyme cascade reactions. To fully understand the mass transport regulating kinetics of enzyme cascade reactions, we investigate the contribution of convective and diffusive transports to a cascade reaction of β-Galactosidase (β-Gal)/Glucose oxidase (GOx) confined in a microchannel. The β-Gal and GOx are respectively assembled on two separated gold films patterned in a polydimethylsiloxane (PDMS) microchannel with a controllable distance from 50 to 100 μm. Experimental results demonstrate that both the decrease in distance of enzyme cascade and increase in flow rate of substrate will accelerate the enzyme cascade reaction. Together with the simulation results, individual reaction kinetics of enzyme cascade reaction can be extracted. We find that the reaction rate catalyzed by β-Gal occurs much faster than by GOx, and thus, the β-Gal catalytic reaction shows diffusion controll while the GOx catalytic reaction shows kinetic controll. Due to the faster local fluid velocity in the gap region between two enzymes than the ones in the enzymes regions, the intermediate glucose generated from the first enzymatic reaction (β-Gal) will be rapidly transported to the second enzymatic reaction (GOx), thus, the enzyme cascade reaction yield will increase with decreasing the gap distance. This phenomenon is similar to the intermediates pool of tricarboxylic acid (TCA) cycle in the metabolic system. This work would promote the understanding of the metabolic/signal transduction processes and active transport in biological systems, and promise to design high performance biosensors and biofuel cells system.