In bone tissue engineering, a qualified scaffold should provide a three-dimensional porous structure mimicking the extracellular matrix of bone except good biological properties. In this study, biomorphic silk fibroin (SF) scaffolds were fabricated from cane by an innovative biotemplating-negative mold process. The physicochemical properties, in vitro enzymatic degradation behavior and biocompatibility of the biomorphic SF scaffolds were investigated. The results showed that the scaffolds well inherited the original porous morphology of cane, and possessed good mechanical stability. The scaffolds had a compressive modulus of 1.56 ± 0.08 MPa and a porosity of 82.73%, and exhibited a bimodal pore size distribution (15 and 172 μm). The degradation ratio of the SF scaffolds increased with prolonging degradation time and reached 29% within 21 days when exposed to 1.0 U/mL collagenase IA. The in vitro cytocompatibility evaluation indicated that the scaffolds could support cell attachment, proliferation, and osteogenic differentiation of osteoblast-like MC3T3-E1 cells, as assessed by SEM, fluorescent staining, MTT, and ALP activity assays. The results indicated the potential of biomorphic SF scaffolds for bone tissue engineering.