Bone Morphogenetic Protein 2 (BMP-2) is a member of the transforming growth factor superfamily. It plays an important role in stimulating osteoblast differentiation and bone formation, and has been widely utilized in clinical bone repairing by implantation. In this study, the nano-hydroxyapatite (nHA)/silk fibroin (SF) porous scaffolds were fabricated for the sustained delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2), and then used to address the hypothesis that rhBMP-2 delivered from the scaffolds could enhance the bone formation in vitro. We optimized an effective method using a prokaryotic expression system to produce rhBMP-2. The rhBMP-2 was expressed, purified and renatured in vitro. And then the rhBMP-2 was loaded onto the nHA/SF scaffolds. The bioactivities of rhBMP-2-loaded nHA/SF scaffolds were assessed in vitro. The results showed that the rhBMP-2 promoted the osteoblasts adhesion and proliferation on the nHA/SF scaffolds. Also, the rhBMP-2 released from the nHA/SF scaffold stimulated a significant increase in alkaline phosphatase (ALP) activity of osteoblasts in vitro. These results demonstrated that the rhBMP-2-loaded nHA/SF scaffolds could promote the bone regeneration and showed potential applications in the bone tissue engineering.