The kinetic studies on air oxidation of glucose catalyzed by free and immobilized glucose oxidase were carried out in the gluconate buffer solution prepared to develop an efficient production of calcium gluconate crystals. The optimal pH, temperature and gluconate concentration as well as the kinetic parameters in the Michaelis-Menten rate expression were determined for the free enzyme reaction in an airtight batch reactor. The fine manganese dioxide particles were entrapped together with glucose oxidase within the calcium alginate gel beads to decompose hydrogen peroxide produced in the oxidation. The various gel beads containing different concentrations of the enzyme and/or manganese dioxide particles were prepared. The intrinsic kinetics for the immobilized glucose oxidase with no peroxide inhibition were assumed to be the same as those for the free enzyme. The liquid–solid mass transfer around the gel beads suspended in the airtight reactor as well as the competitive inhibition effect of hydrogen peroxide were taken into account for the gel beads. A method to determine the effectiveness factor α and hydrogen peroxide inhibition constantKI for the immobilized glucose oxidase was proposed based on the observed time course of dissolved oxygen concentration. The values of the constant KI were found to be almost equal to those of the Michaelis constantKM. The rate constant for hydrogen peroxide decomposition kP as well as the corresponding effectiveness factor α were also determined from the time course of hydrogen peroxide concentration in a separate batch reactor. Both determinations proposed were verified by comparing the observed prolonged time courses of the dissolved oxygen and hydrogen peroxide concentrations in the reactor with those calculated by solving simultaneously the kinetic and mass transfer equations with the parameters obtained.