Aim: This paper aims to study in-vitro and in-vivo evaluation of chitosan (CHI) biocomposite of gentamicin nanoparticles (GNPs) for wound healing. Methods: In this study, CHI nanoparticles (NPs) were prepared using the ionic gelation technique. GNP biocomposites were examined on the excision wound model in Wistar rats to determine the in-vivo efficiency. Results: The diameter and zeta potential of NPs were between 151-212.9 nm and 37.2 - 51.1 mV, respectively. The entrapment efficiency was in an acceptable range of 36.6-42.7% w/w. The release test information was fitted to mathematical models (Zero, First order, Higuchi, and Korsmeyer-Peppas), and according to calculations, the kinetics of drug release followed the Korsmeyer-Peppas model. A comparison of thermograms revealed that the drug was present in the formulation in a non-crystalline form. Conclusion: Histological studies of the wound showed that the rate of skin tissue repair was higher in the GNP biocomposite treatment group than in the others.