BMP-2 (bone morphogenetic protein-2) has been used in promoting bone formation and, in combination with various delivery carriers, in bone regeneration or bone tissue-engineering practice. In the present study, BMP-2 was immobilized directly on a GBR (guided bone-regenerative) membrane surface made of chitosan nanofibres, providing a bioactive surface that can enhance bone-regeneration capacity. The BMP-2-conjugated membrane surface retained bioactivity for up to 4 weeks of incubation, as well as holding over 50% of the initial BMP-2 attached. The BMP-2-conjugated surface increased osteoblastic cell attachment in accordance with the amount of BMP-2 conjugated. In addition, the BMP-2-conjugated chitosan membrane significantly promoted cell proliferation, alkaline phosphatase activity, as well as calcium deposition when compared with BMP-2-adsorbed membrane, indicating a stable localization of BMP-2 at the surface of the chitosan membrane. Taking these findings together, the conjugation of BMP-2 on a chitosan nanofibrous membrane would seen to be applicable for inducing significant and localized bone formation in future GBR procedures.