Abstract：Objective:To establish a candidate reference method for the determination of glucose in human serum based on high performance liquid chromatography (HPLC). Method:D-galactose as the internal standard was added to the serum samples, and glucose standard was also added to equilibrate. The samples were treated with anhydrous ethanol to remove proteins by precipitation. Glucose and galactose reacted with 1-phenyl-3-methyl-5-pyrazolone in serum under weakly alkaline condition and the derivatives were analyzed by high performance liquid chromatography (HPLC). The concentration of glucose in serum was quantified by a calibration curve. Results:The methodology features of the established HPLC method for determination of serum glucose were evaluated and the results were as follows. The withinrun, betweenrun and total coefficients of variation ranged from 0.33% to 0.67%, 0.02 % to 0.85% and 0.47% to 1.03%, and the averages were 0.51%, 0.38% and 0.68%, respectively. The analytical recovery rate ranged from 98.22% to 101.96%. The average bias of certified reference material SRM 965 was -0.072%, ranged from -0.928% to 0.347%. Conclusion:The established HPLC method for serum glucose determination in this study is accurate, precise and practicable, and may be used as candidate reference method for determination of glucose in serum.