Background: Cell-free bone tissue scaffolds show significant potential for bone repair applications. This study aimed to enhance the bone regeneration performance of synthetic poly(ε-caprolactone) (PCL)-based nanofibrous scaffolds by incorporating natural decellularized bone extracellular matrix (dB-ECM) into the scaffold. Methods: The PCL/dB-ECM nanofibrous scaffolds were fabricated by electrospinning. A series of in vitro and in vivo experiments were conducted to verify the biocompatibility of the PCL/dB-ECM nanofibrous scaffolds, as well as their ability to promote osteogenic differentiation of rabbit bone mesenchymal stem cells (rBMSCs) and to repair bone defects. Results: The incorporation of dB-ECM significantly enhanced the bioactivity of the PCL-based scaffold, yielding superior rBMSCs attachment and proliferation, cytoskeleton extension, and osteogenic differentiation. Furthermore, in vivo assays revealed that the PCL/dB-ECM (2:1) scaffold facilitated new bone formation, with improved trabecular structures and better integration with the surrounding tissues. Conclusions: The PCL/dB-ECM (2:1) nanofibrous scaffold presents a promising strategy for bone defect repair, offering enhanced bioactivity and effective integration with host tissues.