Abstract: An isotope dilution-gas chromatography-mass spectrometry method for the determination of lactic acid concentration in serum was established, and the uncertainty of the measurement results was evaluated. Sodium lactate isotope label was added to the serum sample as internal standard. After pretreatment and silanization derivatization, samples were detected by gas chromatography-tandem mass spectrometry in selected ion monitoring mode. The established quantitative analysis method was used to analyze five different concentrations of lactic acid in serum. The results showed that the method had good linearity, and the correlation coefficient was r>0.999. The average spike recovery rate was 101.2%～102.6%, and the relative standard deviation was 0.16%～0.59 % (n = 6). The concentration of lactic acid in five different serum was 1.16 mmol/L, 2.01 mmol/L, 5.50 mmol/L, 10.02 mmol/L, and 15.63 mmol/L, and the related expanded uncertainty U_rel of the results was less than 3.0% (k = 2). The method had good repeatability, high accuracy, reliable results, which could be used for accurate detection of lactic acid concentration in clinical serum sample.