Many scaffolds are candidates for use in tissue engineering approaches for the repair or replacement of bone defects. Among the scaffolds tested for tissue engineering of bone, bio-derived compact bone scaffold (BDCBS) containing mineralized collagen fibers, phosphorus and calcium, as natural bone does, is one of the most promising candidates for this purpose. To analyze how appropriate the BDCBS would be for tissue engineering purposes, we established an in vitro characterization system to describe the surface properties and cytocompaibility of the scaffold. Surface properties were determined by means of scanning electron microscope and scanning probe microscope. The surface phase was examined with the Fourier transform infrared spectroscopy and X-ray diffraction. Osteoblasts from human embryos were isolated from the periosteum. After in vitro expansion, cells were cultivated on the BDCBS. Real-time cell culture was used to monitor the growth process of cells seeded on the scaffold. Using this in vitro characterization, we were able to demonstrate effective growth of osteoblasts on this scaffold. In summary, BDCBS has the surface characterization similar to a natural bone and also has strong affinity for osteoblast attachment and proliferation, indicating the potential as an effective scaffold used in tissue engineering bone.