Immobilized papain was used to prepare chitosan oligomers and the optimum conditions were as follows: NaAc-HAc buffer system, pH 4.0, temperature 45degreesC. After 24 h reaction with stirring, nanofiltration and ultrafiltration were used to desalt and isolate chitosan oligomers into different molecular weight fractions respectively. The yield of chitosan oligomers with molecular weight under 10,000 was 49.55% to total substrate, while that of between 600 and 2000 was 11.07%. The fractions O-1, O-2 was analyzed by Sephadex G-50M gel filtration chromatography and flight mass spectrograph was employed to analyze the fraction O-3. The results showed immobilized papain could depolymerize chitosan successfully, and separating the product by ultrafiltration membrane was feasible. Further study revealed that the immobilized papain could be repeatedly used at least 10 times. An amount of 1 mg free papain and immobilized papain could produce 2.57 and 17.34 mg chitosan oligomers, respectively. The results indicated that the depolymerization efficiency of immobilized papain was obviously higher than that of free papain.