Two impurities of d-biotin are synthesized:di[3-[(3aS,4S,6aR)-2-oxohexahydrothienoimidazol-4-yl]propyl]acetic acid,4-[(3aS,4S,6aR)-2-oxohexahydrothienoimidazol-4-yl]butane-1,1-dicarboxylic acid.Structure of target compounds are confirmed by 1HNMR.This work further improve the research on the impurities of d-biotin,and can be applied to the quality control and quality assurance of d-biotin.