Guided bone regeneration is an effective method that can enhance bone volume at a defect site of the mandible before material implantation. Layer-by-layer electrospun membranes of polyurethane/silk fibroin (SF) were fabricated to mimic oral soft tissue. The electrospun polyurethane fibers were initially fabricated into a membrane. Next, the polyurethane layer was covered with electrospun SF fibers at different thicknesses. Then, the SF layer was covered with electrospun polyurethane fibers. Afterward, the morphologies of the membranes were observed and analyzed by scanning electron microscopy. The physical properties of the membranes were evaluated from the contact angle and mechanical properties. The biological performances were evaluated by observing cell adhesion, viability and proliferation, alkaline phosphatase activity, and calcium content. The results demonstrated that the membrane with a thin SF core showed better physical properties and mechanical performance than the thicker SF cores. Finally, the results deduced that the membrane with a thin SF core was promising for guided bone regeneration.