This is a report on the encapsulation amoxicillin (AMX) in the N-2-Hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) and N,O-carboxymethyl chitosan (CMCS) nanoparticles (NPs) for biomedical applications. The N-2-HACC/CMCS NPs have broad-spectrum antibacterial properties. In order to achieve sustained and slow drug release, improve drug transport efficiency and bioavailability, prolong drug residence time, and reduce pollution, we synthesized highly efficient, easily absorbed and rapidly degradable nano-formulation veterinary antibiotics in this study. The N-2-HACC/CMCS NPs were used for the encapsulation of AMX, and the cytocompatibility, in vitro release, in vivo drug release kinetics and antimicrobial activity of N-2-HACC/CMCS/AMX NPs were investigated. The NPs displayed a round shape and smooth surface, and the NPs allowed the sustained release of AMX at a much slower rate than that of non-coated AMX. The NPs exhibited excellent cytocompatibility and the antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Streptococcus pneumoniae and Staphylococcus aureus. Moreover, the NPs could store at 4 °C, -20 °C and 25 ± 5 °C for 30 d. These results suggested that the N-2-HACC/CMCS NPs could be availed as a candidate for drug delivery carrier to achieve sustained and slow release, improve bioavailability, prolong residence time at the target site, and reduce the dosage of drug.