This work investigated the preparation of chitosan nanoparticles used as carriers for immobilized enzyme. The morphologic characterization of chitosan nanoparticles was evaluated by scanning electron microscope. The various preparation methods of chitosan nanoparticles were discussed and chosen. The effect of factors such as molecular weight of chitosan, chitosan concentration, TPP concentration, and solution pH on the size of chitosan, nanoparticles was studied. Based on these results, response surface methodology was employed. The results showed that solution pH, TPP concentration, and chitosan concentration significantly affected the size of chitosan nanoparticles. The adequacy of the predictive model equation for predicting the magnitude orders of the size of chitosan nanoparticles was verified effectively by the validation data. Immobilization conditions were investigated as well. The minimum particles size was about 42 +/- 5 nm under the optimized conditions. The optimal conditions of immobilization were as follows: one milligram of neutral proteinase was immobilized on chitosan nanoparticles for about 15 min at 40 degrees C. Under the optimized conditions, the enzyme activity yield was 84.3%.