Abstract： Objective To prepare the human bone sialoprotein (BSP) monoclonal antibodies (mAb)with high titer and specificity and identify its characterization, which is based on further studying BSP as clinical biomarker for breast cancer metastasizing to bone. Methods BALB/c mice were immunized with purified recombinant BSP protein. Cell fusion was performed between mouse splenic cells and myeloma cells (Sp2/0), and then the hybridoma cell lines secreting mAb against BSP antigen were screened and cloned. The ascites were prepared and purified with Protein G affinity chromatography. The titer and subtypes of mAb against BSP were identified and measured by ELISA and Western blotting analysis. Results Nine hybridoma cell lines that stably secreted mAb against BSP were successfully obtained. Two of them, D001 and D002,were further identified, which belonged to the subtypes of IgG1 and κ light chain. The two antibodies titers in culture supernatant were 1∶5120 and 1∶10 240, respectively, and those in the ascites fluid were 1∶25 600 and 1∶51 200, respectively. Results of Western blotting analysis and immunohistochemistry showed that the two antibodies could specifically bind with BSP derived from human breast cancer cells. Conclusion Nine mAb against BSP have been successfully prepared which can be used for further studying the biological properties of BSP and reveal its relationship with data from clinic patients.