Chitosan is a natural polymer that can be modified into chemically different types. In order to imply biomedical applications, the chitosan dissolved in aqueous medium. Therefore, in the present study, functionally substituted chemical groups such as O-carboxymethyl chitosan (CMC), N-succinyl chitosan (NSC), quaternary ammonium chitosan (QAC) and tripoly phosphate chitosan (TPC) were synthesized. The peptide derived from Clarias batrachus was used as a drug that is successfully entrapped with NSC nanoparticles and were characterized by FTIR, AFM, SEM, Zeta sizer, fluorescent imaging and SDS-PAGE. In addition, antimicrobial activity was examined. The results of NSC obtained particle size (~64 nm) with highest solubility and 80% of antimicrobial activity against multidrug resistant pathogen Klebsiella pneumonia. Finally, the release profile indicates that the NSC nanoparticles deliver the peptide with 75% sustainable release and good availability up to 96 h. The current research was the first to report the characterization of peptide with NSC nanoparticles by SDS-PAGE, in which the antimicrobial activity was enhanced up to 80%, the particle sizes were obtained ~64 nm from SEM and AFM, and NSC-peptide exhibited 75% of sustainable release and good availability up to 96 h.