Nanotechnology-based fabricated wound dressings are known as appropriate substrates to enhance healing in both acute and chronic wounds. These types of materials have the ability to deliver therapeutic agents. In this study, a wound dressing including heparinized zinc oxide nanoparticles in combination with chitosan and poly(vinyl alcohol) was developed to investigate its antibacterial and regenerative properties in a rat model of full thickness skin wounds. By adding nanoparticles, the mechanical strength increased up to twice as compared to the sample without nanoparticles. In addition, heparin release profile follows the Hixson-Crowell release kinetic. Protein adsorption enhanced by adding nanoparticles in hydrogels and the prepared wound dressings were completely biocompatible. In terms of antibacterial activity, the minimum inhibitory concentration decreased by conjugation of heparin on the surface of zinc oxide nanoparticles compared to the non-functionalized nanoparticles, and, this shows the increased antibacterial synergistic effect by adding heparin to nanoparticles. Furthermore, it was found that the heparinized zinc oxide nanoparticles effectively accelerate wound closure, re-epithelialization and decrease collagen deposition compared to other groups after implantation. Hence, the prepared wound dressings have the capacity to significantly enhance healing of acute wounds.