OBJECTIVE: To investigate the preparation of bone acellular extra-cell matrix(AECM) and to analyze its component. METHODS: With low-osmosis theory and method of cell extraction by detergent, bone acellular extra-cell matrix was prepared. We observed morphologic changes with HE, Mallory-Heidenhain rapid one-step dyeing and Alcian blue dyeing and examined fibronectin(FN) and laminin(LN) with immunohistochemistry. RESULTS: Light microscope showed that the collagen fibers arranged regularly in AECM with blankness of bone lacunas by HE, Mallory-Heidenhain rapid one-step dyeing and that the region around bone lacunas was stained different degrees of blue-green by Alcian blue dyeing. The result of immunohistochemistry showed there are positive markers of FN and LN in ECM. CONCLUSION: This method for preparation of bone acellular extra-cell matrix is effective, and it can keep natural structure of collagen fibers and maintain components of ECM, such as proteoglycan, FN and LN.