Bone tissue has the potential to self-heal and renew, but large-scale bone abnormalities caused by infection, trauma, or any malignancy shall require intervention from external sources. To mimic the host tissue i.e., bone mechanical properties, the scaffolds for bone tissue engineering are designed. However, the host tissue mechanical properties are anatomic site-specific as per the location where a scaffold has to be implanted to facilitate tissue regeneration. This is because of the heterogeneous and hierarchical structure of bone that consists of mineral nano-particles, water, collagenous protein, etc. which makes bone a more complex material. Fabricating nano-scaffolds becomes a challenging task that can resemble the bone properties in form of biological, chemical, or mechanical. As a result, it is required to examine the change in mechanical properties of a bone throughout the site to build efficient nano-scaffolds for tissue engineering/bone regeneration. The mechanical characteristics of cortical bone were investigated at three diaphysis regions of the femoral cortical bone under tension and compression stress. The obtained mechanical properties have been compared statistically using a t-test at p < 0.05 and found a statistical difference in the mechanical properties for different diaphysis locations. The findings of this research may be helpful for the researchers working on the design of nanoparticle-based composite biomaterials to mimic the anatomic site-specific mechanical properties of bone for tissue engineering applications.