Beta 2-microglobulin (B2M) is a commonly used detection index in clinical laboratories. Currently, it is used as a sensitive indicator for the early detection of kidney disease. Immunoassay is the most commonly used method for B2M detection in clinical practice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has changed the face of laboratory testing by providing high-throughput analysis with better specificity than detection methods using only antibodies. An isotope-dilution LC-MS/MS (ID-LC-MS/MS) method was developed for serum B2M quantification. After B2M denaturation, reduction and alkylation of cysteine residues, trypsin was added for digestion of B2M. Then, it was purified by a solid-phase extraction column, and the sample was injected into a high-performance LC-MS/MS for measurement. A signature peptide (VNHVTLSQPK) was selected as a surrogate for B2M. A stable isotope-labeled peptide (VNHVT[13C615N]LSQP) was used as the internal standard to quantify B2M based on the calibration curve method. The linear range of serum B2M calibration curve was from 0.25 to 40 mg/L. The limit of quantification was 0.25 mg/L, and limit of detection was 0.06 mg/L. At different concentrations of serum B2M, the precision (coefficients of variation, CV%) ranged from 1.47% to 3.97%, and accuracy (relative error, RE%) was within -3.15% and 6.80% of nominal values. The applicability and reliability of the method were verified by measuring B2M in the ERM-DA470k/IFCC and serum samples. The validated LC-MS/MS method was successfully applied to a clinical study involving quantification of serum B2M in patients with acute renal insufficiency and healthy individuals. Deming analysis showed that the ID-LC-MS/MS and immunoassay were in good agreement. The LC-MS/MS method we developed is sensitive and reliable for the absolute quantification of serum B2M.