Bone tissue engineering necessitates a three-dimensional porous scaffold design to closely mimic the in vivo physiological biomimetic microenvironment with good mechanical properties to support the adhesion and proliferation of osteoblasts. In this study, a silk fibroin/cellulose nanowhiskers-chitosan (SF/CNW-CS) composite scaffold with excellent mechanical properties and biological compatibility was prepared by layer-by-layer assembly of CNW and CS onto a porous SF scaffold fabricated by freeze-drying. The morphology, assembly construct, and mechanical properties of the SF/CNW-CS composite scaffold were characterized using different technical methods. The hierarchical lamellar structure was formed by assembly of CNW and CS onto the SF lamella. Increasing the number of CNW and CS assembly layers to 108 slightly decreased the porosity from 86 to 78 % and simultaneously changed the compressive stress-strain curves from soft to hard tissue-like properties with an increase in compressive modulus from 2.5 to 12.5 MPa. Human MG-63 osteosarcoma cells were further cultured on the SF/CNW-CS scaffold to evaluate their suitability for bone tissue engineering. The results indicated that the SF/CNW-CS composite scaffold supported cell proliferation and promoted the levels of biomineralization-relevant alkaline phosphatase activity and osteocalcin expression over those for a porous SF scaffold. Taken together, our results indicate that the porous SF/CNW-CS composite material will be a promising tissue engineering scaffold for bone generation and implantation.