Background: In order to ensure the accuracy of the product, we established 1st model of metrological traceability hierarchy for light-initiated chemiluminescent assay (LICA) of 17 beta-estradiol (E-2) at the manufacturer, based on International Organization for Standardization (ISO) 17511:2020. Moreover, we verified/validated the basic performance (such as matrix effect and long-term stability of end-user IVD MD calibrator, precision, linearity interval, accuracy/ trueness, and detection capability) at the clinical end-user. Methods: Human serum samples were used in this study. E-2 was detected by mass spectrometry (MS) and LICA. The metrological traceability of LICA for E-2 was established according to ISO 17511: 2020 standards, and pools of human samples were used as the m.3. secondary calibrator. Precision was validated according to Clinical and Laboratory Standards Institute (CLSI) EP05-A3. The linear interval was verified according to CLSI EP06-ED2. Comparison of accuracy and trueness of E-2 with MS and Roche according to CLSI EP09-A3. The detection capability was validated according to EP17-A2. Matrix effect and long-term stability evaluation of end-user IVD MD calibrator were carried out according to CLSI EP14-A2, EP25-A. Statistical software was used for data analyses. Results: The use of pools of human samples and fine adjusting calibrators ensured the accuracy of end-user test results. The metrological traceability of LICA for E-2 was established. It showed excellent precision, meeting the requirements of allowable imprecision (7.5%). The allowable deviation from linearity (ADL) of 5% was allowed to show a good linear interval (12.52-4167.25 pg/ml). The accuracy/ trueness was verified, and relative deviation in the medical decision level met the performance specification of 10.03% compared with MS or Roche. The validated limit of blank, limit of detection, and limit of quantitation of E-2 were 4.95 pg/ml, 8.93 pg/ml, and 9.88 pg/ml, respectively (the allowed imprecision is 20.00%). The interference rate of E2 ranged from -5.5% to 6.6%. Conclusion: LICA showed high sensitivity, high specificity, excellent precision, wide linearity interval, IVD MD calibrator has long-term stability, and no matrix effect. The metrological traceability of E-2 established by using pools of human samples as M.3. can deliver accuracy to the end-user IVD MD and show good consistency with MS and Roche.