Abstract: The relationship of free amino groups on the surface and the characteristics of chitosan nanoparticles (CS-NPs) prepared by ionic gelation method was investigated. Free amino groups on the surface of CS-NPs were determined by colloidal titration, and the effects of the amount of free amino groups and its ionizable level on the particle size, zeta potential, appearance, drug entrapment efficiency and drug release profile in vitro of CS-NPs were investigated. The result showed that the surface free amino groups reduced, the average size, zeta potential, stability of nanoparticles, and the drug release rate and degree all decreased while the drug entrapment efficiency was not affected with the increase of tripolyphosphate (TPP) concentration. With the increase of pH, the free amino groups could be deprotonated and the ionizable level was stepped down, correspondingly the particle size and zeta potential of CS-NPs decreased. Additionally, the drug release rate and degree were elevated in acid medium while descended in neutral or base medium. The amount and ionizable level of free amino groups on the surface are affected by the gelation degree and pH, which further affected the volume phase transitions (swelling / shrinking processes) of CS-NPs. The properties of CS-NPs have correlation with the surface free amino groups.