Abstract Chitosan is a linear polysaccharide with and prominent physicochemical and biological properties such as biocompatibility, biodegradability, nontoxicity, non-immunogenicity, bioadhesion, antibacterial, antifungal and hemostatic activity. Due to these properties, it has found many applications in cosmetic, textile, and food industries; agriculture; biotechnology; pharmaceutical industry and medicine; specially in biomedical applications. The special chemical structure of chitosan allows some specific modifications and by reducing the size of chitosan particles to Nano size, it becomes an excellent drug nanocarrier. Vancomycin is a typical antibiotic used for bacterial infections caused by geram positive bacteria. In this work, chitosan nanoparticles (CSNPs) were prepared via ionotropic gelation using tripolyphosphate (TPP) as crosslinker. The effect of chitosan and TPP concentration on the size of chitosan nanoparticles was studied and CS:TPP ratio of 1:1 with average size of nanoparticle about 100 nm were selected. The prepared samples were characterized using DLS, FTIR, TGA, DSC, and SEM techniques. The results confirmed that vancomycin has been loaded successfully on chitosan nanoparticles and there was not any interaction between vancomycin and chitosan. Also, it is observed that 40% of vancomycin is released burstly in the first 9 h and after that the drug release is continued gradually to receive 90% at 100 h.