The guided tissue regeneration (GTR) membrane is a barrier intended to maintain a space for alveolar bone and periodontal ligament tissue regeneration but prevent the migration of fast-growing soft tissue into the defect sites. This study evaluated the physical properties, in vivo animal study, and clinical efficacy of hydrophilic PLA95/β-TCP GTR membranes prepared by electrospinning (ES). The morphology and cytotoxicity of ES PLA95/β-TCP membranes were evaluated by SEM and 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) respectively. The cementum and bone height were measured by an animal study at 8 and 16 weeks after surgery. Fifteen periodontal patients were selected for the clinical trial by using a commercial product and the ES PLA95/β-TCP membrane. Radiographs and various indexes were measured six months before and after surgery. The average fiber diameter for this ES PLA95/β-TCP membrane was 2.37 ± 0.86 µm. The MTT result for the ES PLA95/β-TCP membrane showed negative for cytotoxicity. The significant differences in the cementum and bone height were observed between empty control and the ES PLA95/β-TCP membrane in the animal model (p < 0.05). Clinical trial results showed clinical attachment level (CAL) of both control and ES PLA95/β-TCP groups, with a significant difference from the pre-surgery results after six months. This study demonstrated that the ES PLA95/β-TCP membrane can be used as an alternative GTR membrane for clinical applications.