A highly accurate liquid chromatography tandam mass spectrometry (LC-MS/MS) based candidate reference measurement procedure (cRMP) for the determination of human serum 17 beta-estradiol (E-2) was developed. A bracketing calibration method (BCM) based on internal standard (IS) concentrations for each sample used to best match the analyte concentration was compared to the classical calibration curve-based method with a constant IS concentration. Precision was analyzed according to the inter-assay reproducibility (n = 15) and intra-assay repeatability (n = 9). Accuracy was confirmed using certified reference materials (CRMs) and further validated by comparison to the established RMPs. Sixty patient serum samples were collected, and the E-2 levels were measured by the BCM isotope dilution (ID) procedure coupled with the LC-MS/MS method to evaluate three immunoassays commonly used in clinical laboratories in China. Limits of quantification (LoQ) as low as 5 pg/mL (18 pmol/L) were achieved. The isomer of E2 (17 alpha-estradiol) was successfully separated by optimizing the LC method. The results of method validation showed that the intra- and inter-assay imprecisions were 1.84 similar to 2.91% vs 2.08 similar to 9.53%, while the analytical recoveries were 98.73 similar to 100.77% vs 93.72 similar to 102.39% for the BCM and classical calibration curve quantification method, and the BCM demonstrated a higher agreement with CRMs and established RMPs. Bland-Altman plots of the E, results revealed concentration-dependent biases. In conclusion, a precise, facile, and reliable ID-LC-MS/MS method is developed in this study. It is demonstrated that the use of the BCM could achieve higher precision and accuracy, which better meet the needs of RMPs.