In this study, blends of chitosan (CS)/silk fibroin (SF) and reduced graphene oxide (rGO) as guided bone regeneration membranes were fabricated by solvent casting method and their architectural features, hydrophilicity, porosity, swelling, degradation, and mechanical properties were investigated. The influence of CS, SF, and rGO on osteoblasts spreading and attachment was also determined by scanning electron microscopy (SEM) images and DAPI (4', 6-diamidoino-2-phenylindole) staining assay. The osteogenic differentiation was determined by the alizarin red staining and alkaline phosphatase activity. The results demonstrated that, the hydrophilicity, swelling and degradability decreases with the increase of SF content, whereas tensile strength increases accordingly. It was confirmed that with increasing the rGO concentration, the porosity and tensile strength decreased but the hydrophilicity was improved. The cell behaviors of G-292 cells were enhanced by increasing the CS content. According to the results, it can be concluded that, SF/CS/rGO blended membranes are promising candidates for bone tissue engineering and optimum results were obtained for the membrane composed of SF:CS: rGO with 84:7:9 weight ratio. It should be noted that the optimized membranes should be further studied for clinical applications.