Objective To develop a reference measurement procedure for the determination of human serum homocysteine by isotope dilution high performance liquid chromatog.-tandem mass spectrometry (ID-LC/MS/MS), and to create the routine measurement system of BSBE tracing to the reference measurement procedure, which could ensure the accuracy of the routine measurement system. Methods We selected conditions for chromatog. and mass spectrometric, and conducted pretreatment optimization, then established a homocysteine ID-LC/MS/MS reference measurement procedure. We established HCY traceability system according to ISO17511, and ensured the routine measurement system of BSBE was able to trace to the reference measurement procedure. Results The linear range of homocysteine in serum samples was determined to be 1-80μmol/L. RSD of repetitive precision and reproducibility precision were less than 1.72% and 2.90%, resp. The bias of measurement of SRM1950 and SRM1955 was less than 0.82% and 1.81%, resp. The correlation coefficient R2 of the reference measurement procedure and the selected measurement procedure calibrated by the working calibrator was 0.9952, with the slope of 1.009. The correlation coefficient R2 of the selected measurement procedure calibrated by the working calibrator and the standard measurement procedure calibrated by the working calibrator was 0.9984, with the slope of 1.005. Conclusion ID-LC/MS/MS method which is to determine the homocysteine reference measurement procedure in human serum is successfully established in the current study, and we also establish the traceable chain of HCY to ensure the accuracy of the measurement results of BSBE.