Objective To investigate the expression of osteoblasts bone sialoprotein(BSP) and evolutionary trace. The mature peptide gene sequences were analyzed to obtain BSP modification sites. Methods Total RNA was isolated from cultured osteobalsts and desired cDNA fragment was obtained by RT-PCR with two gene specific primers. The segment was inserted into pBluescript KS vector and the result plasmid was transformed into DH5α. The positive clone and sequence were performed. Using BSP as seed sequences, the searching of BSP gene sequence and its homologous protein with various bioinformatics tools were retrieved from NCBI database. The comparative studies were taken for BSP evolutionary trace sites and related functional sites. Results There was a complete structural domain of 72 homologous protein sequences, 16 bone sialoprotein family BSP-propeptide domain, 10 RGD binding domain and the BSP domain 33 subfamily specific residues. The ligand of bone sialoprotein RGD domain binding sites was mainly distributed in the middle region with pockets. 900bp specific fragment was obtained. The sequence of the gene encoding BSP mature pepide coincided with that of the references published. Conclusion The gene encoding BSP mature peptide is obtained from osteoblasts that express and translate into active bone sialoprotein. There are methylation and acetylation sites existed on the BSP mature peptide. This result will help us to further investigate the expression and function of BSP.