Characterizations of chitosan nano-particles prepared by the ionization gelation method were discussed in this paper. The optimal pH of an immobilized enzyme was 7.5. The optimal temperature of the immobilized enzyme was 45 °C. The thermal, operational and storage stabilities of an enzyme were improved after they were immobilized on chitosan nano-particles. The chitosan nano-particles were applied to protect the activity of free neutral lipase. They could improve the enzyme activity by 13.17% than that of the free neutral lipase. The Michaelis constant K m of the immobilized enzyme was disclosed as 0.37×10 2 g/l by the Line-weaver Burk plot at 40 °C.