The three-dimensional functional nanofibrous microenvironment plays a key role in the regulation of cellular growth, because it can mimic the natural extracellular matrix structure. As an important nanofibrous scaffold, 3D functional nanofibers display broad application prospects in biomedical areas, such as tissue engineering, drug release, and biosensors. Recently, these functional nanofibrous scaffolds based on biocompatible natural materials with excellent flexibility and biochemical characteristics show great potential in tissue engineering applications. Herein, we prepared 3D chondroitin sulfate surface-modified silk nanofibers (3D CS-SNFs) using a combination of electrospinning and chemical grafting methods. The as-prepared 3D CS-SNFs exhibited excellent biocompatible properties. Moreover, there are numerous deeply interconnected pores (larger than 20 mu m) for enhanced cellular entry into CS-SNFs and for 3D cell culture in vitro. The 3D CS-SNFs are beneficial to cell adhesion, nutrient delivery, and excretion of excrement, which provides healthy growth environment and living conditions for those cells that penetrate into the interior of the scaffold. Our results indicate that the 3D CS-SNFs show much higher degrees of promotion of bone marrow mesenchymal stem cells adhesion and osteogenic differentiation, as compared with the 3D raw silk nanofibers. These results demonstrate that the as-prepared 3D CS-SNFs could provide a suitable 3D microenvironment for cellular growth, adhesion, and excellent osteogenic differentiation. This work paves a new way for 3D cell culture construction for damaged bone repair and regeneration.