The preparation and properties of immobilized flavourzyme on globular cross-linked chitosan, with glutaraldehyde as crosslinking agent, were investigated. The optimal concentration of chitosan, NaOH and glutaraldehyde was 2.5%, 3%, and 0.5%, respectively, and the optimal crosslinking time was 6h. The optimal flavourzyme dosage was 40mg/g carrier, and the optimal immobilizing reaction temperature, pH, and time was 35℃, pH 5.5, and 10h, respectively. After immobilized, the optimal reaction temperature of flavourzyme was improved from 50℃ to 70℃, and the optimal pH value was improved from 7.0 to 8.0. Moreover, the ranges of temperature and pH for keeping the higher enzyme activity had been extended. The stability of storage and operation of the immobilized flavourzyme was good enough to be reused for many times, which suggested that the immobilized flavourzyme was suitable for the wide applications in industry.