Regenerative procedures using guided tissue regeneration (GTR) membrane are presently well-established in periodontal therapy. In this study, bilayered poly(lactic-co-glycolic acid) (PLGA)/wool keratin (WK) composite membranes were prepared for use as GTR membrane by the solvent casting and electrospinning methods. Then, the composite membranes were used to evaluate the effects in guided tissue regeneration in beagle dogs. The results showed that the bottom layer (casting film) of the bilayered PLGA/wool keratin composite membranes was a compact PLGA/wool keratin membrane, and the upper layer (electrospun film) was a loose, porous, three-dimensional, and fibrous PLGA/wool keratin membrane (The wool keratin was at five different levels: 0, 0.5, 1, 2, or 4 wt %). The mechanical properties of the composites were significantly enhanced by the addition of wool keratin. The bilayered PLGA/1.0% wool keratin composite membranes presented the best values of ultimate strength and Young's modulus. All of the bilayered PLGA/wool keratin composite membranes showed high thermal and thermooxidative stabilities. The GTR results showed that the PLGA/1.0% wool keratin composite membranes could effectively promote the periodontal tissue regeneration after 12 weeks, and have a similar effect to the collagen membrane on the regeneration of the periodontal tissue. Thus, the bilayered PLGA/wool keratin composite membranes show great potential to meet the demand for GTR membrane. The study will serve as a foundation for further study of the PLGA/wool keratin membranes for GTR application and the therapy of periodontal disease.