Chitosan (CS) nanoparticles have several distinct intrinsic advantages; however, theirin vivocolloidal stability in biological fluids was not fully explored especially when carrying proteins. The present study aimed to investigate their colloidal stability using anex vivophysiological model of fetal bovine serum (FBS) and human serum (HS). The stability of bovine-serum-albumin (BSA-) loaded nanoparticles was relatively higher in FBS than that in HS. Particle size of unloaded and BSA-loaded nanoparticles was statistically unchanged up to 24 h after incubation in FBS. However in HS, a significant increase in particle size from 144 ± 17 to 711 ± 22 nm was observed for unloaded nanoparticles and by 2.5-fold for BSA-loaded nanoparticle, at 24 h after incubation in HS. Zeta potential of both nanoparticles was less affected by the components in FBS compared to those in HS. A remarkable swelling extent was experienced for unloaded and BSA-loaded nanoparticles in HS, up to 54 ± 4% and 44 ± 5%, respectively. Morphology of unloaded and BSA-loaded nanoparticles was varied from smooth spherical and rod shape to irregular shape when incubated in FBS; however, form agglomerates when incubated in HS. These findings therefore suggest that HS is more reactive to cause colloidal instability to the chitosan nanoparticles compared to FBS.