In this article gentamicin (GM) impregnated microspheres were used to extend the drug release time for the treatment of chronic osteomyelitis. The granules were prepared in solution and consisted of nanohydroxyapatite (nHA), chitosan (CS) and GM loaded ethyl cellulose (EC) microspheres. A rabbit model with chronic osteomyelitis was made by using staphylococcus aureus and morrhuate sodium and special inspection methods were used to test the curative effects of the granules, such as microbiological investigations, tissue, and X-ray observations. The granules were provided with excellent drug release properties, 49 days in vitro and 45 days in vivo, moreover, they showed almost no cytotoxic for fibroblast and osteoblast. The findings indicated that the GM-impregnated CS/nHA/EC microspheres granules showed outstanding curative effect. Generally, it can be concluded that the granules containing GM impregnated microspheres may be used effectively in the treatment of the chronic osteomyelitis.