Background: Yunnan Baiyao, as a traditional Chinese herbal remedy with definite curative effect, has recently been proven can promote the proliferation of osteoblasts and differentiation of human periodontal ligament fibroblasts. Bio-Oss (R) scaffold is a porous bone graft material of natural and antigen-free bovine bone origin. Methods: To observe the effect of Yunnan Baiyao on the differentiation of HPDLFs on the Bio-Oss (R) collagen scaffold in vivo, the HPDLFs-Bio-Oss (R) collagen complex was constructed in vitro, and Yunnan Baiyao aqueous solution was added, respectively. The complex was divided into Yunnan Baiyao group I (50 mu g/mL), Yunnan Baiyao group II (100 mu g/mL), positive control (rhBMP-2) group and sham group. HPDLFs were identified by immunocytochemistry and immunofluorescence. The compatibility of HPDLFs with Bio-Oss (R) collagen was observed by fluorescence microscope and scanning electron microscope. The specimens were taken for HE staining after 8 weeks since the complex was implanted into nude mice, and the expressions of osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN), and collagen I (CON-I) were detected by immunohistochemistry and qRT-PCR. Results: The number of capillaries and osteoblasts increased significantly after Yunnan Baiyao stimulation, and the expressions of BSP, OC, OPN and CON-I were increased after Yunnan Baiyao stimulation in a dose-dependent manner. Conclusion: Yunnan Baiyao solution can promote the differentiation of HPDLFs and the generation of capillaries on Bio-Oss (R) collagen scaffold in a dose-dependent manner.