Silk fibroin (SF) and β-tricalcium phosphate (β-TCP) had been used in biomedical applications for these years. The potential of silk and β-TCP for application in tissue engineering is currently being explored. The purpose of this study was to prepare and characterize a 3D scaffold consisting of nano-β-TCP/SF composite. XRD and FT-IR analysis showed that predominant crystalline phase of calcium phosphate was β-TCP; a chelate effect between SF and Ca2+ was happened at complexing period of SF and β-TCP. The compressive strength of nano-β-TCP/SF composite was 42 MPa ± 0.12 MPa. In vitro cell cultivation experiment showed that the composite was a good matrix for the growth of osteoblasts. Conclusion: the incorporation of SF into nano-β-TCP can enhance both mechanical strength and bioactivity of the scaffold, which suggests that the β-TCP/SF composite may be a potential biomaterial for tissue engineering.