Background: The aim of this study is to verify the analytical performance of four homocysteine detection systems made in China and to explore the comparability of homocysteine detection systems by isotope dilution liquid chro-matography-tandem mass spectrometry (ID-LC-MS/MS) reference method. Methods: The intra-batch precision, inter-batch precision, accuracy, and linear range of four homocysteine detec-tion systems were evaluated. The ID-LC-MS/MS reference method was used to evaluate the comparability and ac-curacy of fresh frozen serum samples in four different detection systems of homocysteine. The ID-LC-MS/MS ref-erence method is used to assign samples as calibrators to calibrate each system. The variation and deviation of fresh serum samples between different systems before and after calibration were compared. Results: The intra-batch imprecision of the four detection systems was less than 5%, and the coefficient of varia-tion of inter-batch imprecision was less than 6.7%. The precision met the clinical requirements. Before calibra-tion, the results measured by detection system 2 are consistent with the ID-LC-MS/MS reference method, which meets the requirements of accuracy verification. The regression equation of R ≥ 0.975 in the regression equation of linear analysis of the four systems, the linearity of the four detection systems is good in the range of evaluation concentration, and all of them can meet the declared linear range. The absolute average bias of fresh serum mea-sured by the four detection systems after calibration decreased from 3.76 μmol/L, 0.96 μmol/L, 1.30 μmol/L, -1.56 μmol/L to 0.31 μmol/L, 0.28 μmol/L, 0.4 μmol/L, 0.40 μmol/L, respectively. The relative average bias de-creased from 22.6%, 7.50%, 11.0% and -8.50% to 1.98%, 1.78%, 2.59%, 2.34%, respectively. After calibration, the slope and intercept of the regression curve of the fresh serum measured by the four detection systems and the reference method are closer to 1 and 0 than before calibration. Conclusions: The precision, reference interval, and linear evaluation of the four detection systems are good. The ID-LC-MS/MS reference method assigning fresh frozen serum samples as calibrators can improve the accuracy and comparability of the results of different detection systems. © 2021 Verlag Klinisches Labor GmbH. All rights reserved.