Xenograft bone has been widely used as a bone grafting material because it gains advantages in biological and mechanical properties as compare with the use of an allograft bone. Heat-treatment of bone is recognized as one of the simple and practical methods to lower the human immunodeficiency virus (HIV) infection and overcome the risks of rejection and disease transfer during the bone transplantation. Therefore, understanding the change of bone’s organic matrix after heat treatment has become a significant topic. In this study, thermal gravimetric analysis (TGA) was used to investigate the condition of organic constituents of a bovine cortical bone. In order to well characterize the microstructural and mechanical property of the bone after heat treatment, nanoindention technique was also employed to measure the localized elastic modulus (E) and hardness (H) of its interstitial lamellae and osteons lamellae at the temperatures of 23°C (RT), 37°C, 90°C, 120°C and 160°C, respectively. The TGA results demonstrated that heat-treated bones had three stages of weight loss. The first stage was the loss of water, which started from RT to 160°C. Follow by a weight loss of organic constituents starting from 200°C to 600°C. Upon reaching 600°C, the organic constituents were decomposed and mineral phase loss started taking place until 850°C. From the nanoindentation results, it showed the values of E and H measured for the interstitial lamellae were higher than that of the osteons lamellae. This phenomenon indicates that the interstitial lamellae are stiffer and easy to be mineralized than osteons lamellae. For a specimen heat-treated at 90°C, the values of E and H of interstitial lamellae and osteons lamellae were similar to a non-heat-treated specimen. For a specimen heat-treated at 120°C, its interstitial lamellae had higher E and H values than osteons lamellae. When a specimen was heat-treated at 160°C, both interstitial lamellae and osteons lamellae demonstrated a slight decrease of their E and H values. An ANOVA statistical analysis was used to analyze the difference in elastic properties and hardness in various temperature ranges.