Blood glucose is an important monosaccharide functioning as the main source of energy for the human body. The accurate measurement of blood glucose is crucial for the screening, diagnosis, and monitoring of diabetes and diabetes-associated diseases. To assure the reliability and traceability of blood glucose measurements, we developed a reference material (RM) for use in human serum at two different concentrations, which were certified by the National Institute of Metrology (NIM) as GBW(E)091040 and GBW(E)091043.Raw serum samples were collected from residual samples after clinical testing, filtered, and repackaged under mild stirring. The homogeneity and stability of the samples were examined according to ISO Guide 35: 2017. Commutability was evaluated in compliance with CLSI EP30-A. Value assignment was carried out in six certified reference laboratories using the JCTLM-listed reference method for serum glucose. Moreover, the RMs was further applied in a trueness verification program.The developed RMs was homogeneous and commutable enough for clinical use. They were also stable for 24 h at 2-8 ℃ or 20-25 ℃ and for at least 4 years at - 70 ℃. The certified values were 5.20 ± 0.18 mmol/L and 8.18 ± 0.19 mmol/L (k = 2) for GBW(E)091040 and GBW(E)091043, respectively. The pass rates were evaluated by bias, coefficient of variation (CV), and total error (TE) for 66 clinical laboratories in the trueness verification program were 57.6%, 98.5%, and 89.4% of GBW(E)091040, and 51.5%, 98.5%, and 90.9% of GBW(E)091043, respectively.The developed RM could be used for the standardization of reference and clinical systems with satisfactory performance and traceable values, providing strong support for the accurate measurement of blood glucose.Copyright © 2023 Elsevier B.V. All rights reserved.