Chitosan nanoparticles were prepared by ionic cross-linking with tripolyphosphate (TPP). The major effect on encapsulation and release of protein in chitosan-TPP nanoparticles was investigated in order to control the loading and release efficiency. A set of the same molecular weight (MW) proteins with different pI and a set of the same pI proteins with different MW were studied. The influence of protein concentration, pH of solution, and the activity of released protein were examined. It was found that the encapsulation efficiency (EE) of a set of the different MW protein decreased with increasing of MW of protein and protein concentration. The protein with having pI higher than pH of solution was attracted to the positively charged chitosan, resulting in increasing of EE. The release of protein from the nanoparticles showed that the protein release decreased with increasing of chitosan concentration, high MW protein, low pH, and less swelling of the particle. The released protein in chitosan-TPP matrix was still active in the buffer solution.