Periodontitis is a worldwide chronic inflammatory disease, where surgical treatment still shows an uncertain prognosis. To break through the dilemma of periodontal treatment, we fabricated a three-dimensional (3D) multilayered scaffold by stacking and fixing electrospun polycaprolactone/gelatin (PCL/Gel) fibrous membranes. The biomaterial displayed good hydrophilic and mechanical properties. Besides, we found human periodontal ligament stem cell (hPDLSC) adhesion and proliferation on it. The following scanning electron microscopy (SEM) and cytoskeleton staining results proved the guiding function of fibers to hPDLSCs. Then, we further analyzed periodontal regeneration-related proteins and mRNA expression between groups. In vivo results in a rat acute periodontal defect model confirmed that the topographic cues of materials could directly guide cellular orientation and might provide the prerequisite for further differentiation. In the aligned scaffold group, besides new bone regeneration, we also observed that angular concentrated fiber regeneration in the root surface of the defect is similar to the normal periodontal tissue. To sum up, we have constructed electrospun membrane-based 3D biological scaffolds, which provided a new treatment strategy for patients undergoing periodontal surgery.