The biological mechanism of trabecular bone with its complex three-dimensional microstructure and the interaction between micro-environment and osteoblasts have been studied and applied in the field of bone repair, reconstruction and bone regeneration. However, previous research lack the simulation of the real environment of bone trabecula in vivo, especially the lack of accurate quantification of trabecular microstructure and the research and application of bone regeneration under the condition of interaction with microenvironment. In this study, a new method is used to analyze and construct the three-dimensional environment of trabecular bone. Three-dimensional cancellous bone model and geometric features analysis were conducted by using Mimics and Rhino software based on Micro CT of rabbit proximal femoral cancellous bone scan. Bionic trabecular structure was analyzed and constructed by using Stirling formula and 3D printed trabecular bone structure. The comparison on trabeculae area and structure similarity analysis between cancellous bone samples and bionic cancellous bone show that bionic cancellous bone has extremely high similarity (more than 95%) with natural cancellous bone structure. In vitro experiments of bionic bone trabecular can construct a good microenvironment for cell growth. In sum, the proposed method provides a useful tool for manufacturing a more realistic physiological structure for clinical research and application for tissue engineering.