In this study, glucose and fructose were subjected to catalytic conversion by glucose isomerase in an amino-phase packed column operated using a reverse-phase mobile phase that enabled simultaneous reaction and separation. The mobile phase was composed of acetonitrile and aqueous glucose isomerase solution. The system was used to determine the apparent rate constant, kapp, and the reaction rate constants in the mobile and stationary phases, km and ks, respectively. The static-state reaction kinetic parameters Km and Vm were also investigated. This work has demonstrated that the isomerization of glucose to fructose and the reverse reaction can be studied using appropriate conditions for reaction and separation using a reverse-phase amino column reaction chromatography system.