Analysis of immobilized enzyme in situ is a crucial step to embed an enzyme onto the planar technology of standard integrated circuit (IC) and microelectromechanical systems (MEMS) for a bioreactor or enzyme-coupled biosensor. A surface reaction limited model, based on a systematized and standardized approach, mathematically derived from mass transfer dynamics and the Michaelis-Menten equation for the measuring the apparent K*m (Michaelis-Menten constant) and V*max (maximum reaction rate per unit surface area of catalyst) of an immobilized enzyme on a planar surface was developed. The derived equations for the kinetic model were simulated and experimentally confirmed. A platform of a microflow bioreactor with a one-sided planar catalytic surface that contained immobilized enzyme was constructed. The microfluidic bioreactor was designed to possess a channel height less than that of the diffusion layer thickness in a semi-infinite diffusion process, and K*m and V*max of rat phenol sulfotransferase (PST) immobilized on the silicon oxide surface were successfully determined in situ. Variation in kinetic constants and the possible differences in performance between free and immobilized PST are discussed.