This paper aims to study in-vitro and in-vivo evaluation of chitosan biocomposite of gentamicin nanoparticles (GNP) for wound healing. In this study, chitosan nanoparticles 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. The diameter and zeta potential of nanoparticles were between 151–212.9 nm and 37.2 – 51.1 mV, respectively. The entrapment efficiency was in an acceptable range of 36.6 to 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. 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.