The production of high concentrations of physiologically active pentameric and hexameric chitosan oligosaccharides was investigated. Chitosanase directly immobilized on an agar gel-coated multidisk impeller was used for the hydrolysis of chitosan. As hydrolysis proceeded, the concentration of chitosan was increased from the saturation concentration (20kg/m³) to 50kg/m³ by stepwise addition of chitosan powder with pH control. The timing of the addition of the chitosan powder was determined by monitoring the change in the torque required to agitate the reaction solution. The target products were produced more efficiently when the chitosan powder was added over a short interval rather than a long interval. When hydrolysis of a 50kg/m³ chitosan solution at 50°C was repeated three times using the same immobilized enzyme, the target products were obtained in high concentrations (20kg/m³) from each of the three reactions with no reduction in the activity of the immobilized enzyme.