The purpose of this study was to prepare chitosan nanoparticles (CS NP) for controlled protein delivery. Two techniques, simple ionotropic gelation (method [I]) and w/o/w emulsion solvent evaporation containing ionotropic gelation (method [II]), were used to prepare CS NP. Tripolyphosphate (TPP) and Eudragit L100-55 (Eud) were used as anionic agents to form complex with cationic chitosan. Bovine serum albumin (BSA) was encapsulated into NP. The morphological characteristics, particle size and size distribution, protein entrapment efficiency, zeta potential, in vitro release, protein secondary structure and its integrity were investigated. The results showed that CS NP could be prepared by appropriate cationic and anionic ratios in both methods. Excess anionic agents resulted in particle aggregation of micron size. The median sizes of particles were between 0.127-0.273 mcm with method [I] provided the smallest size. The 0.02-0.10% BSA loaded preparations showed the same particle sizes and size distributions as blank preparations. SEM photomicrographs revealed that the obtained NP were spherical. Protein entrapment efficiency was between 47-84% and increased when decreasing the percentage of drug loading. The method [II] with TPP exhibited the highest protein entrapment efficiency, following by the method [II] with Eud and method [I] with TPP, respectively. The zeta potentials were positive. Prolonged in vitro protein release profiles were observed from all preparations of CS NP. After 10 days, the release was between 53-72%. Circular dichroism and SDS-polyaceylamide gel electrophoresis techniques confirmed that these processes did not have any destructive effect on the protein structure. Therefore these preparation techniques could be used to encapsulate water-soluble drugs, proteins, DNA, or antigens into CS NP as effective delivery carriers.