Abstract: Objective：To establish a chemiluminescence quantitative method for detecting human epididymis protein 4 (HE4), and evaluate its performance. Methods：Serum HE4 can bind with both the anti-HE4 monoclonal antibody coated on micro beads and the anti-HE4 monoclonal antibody labeled with horseradish peroxidase (HRP) to form so-called sandwich structure. Then, the luminescent substrate solution containing luminol, H₂O₂ and enhancer was catalyzed by HRP to produce light signals. According to light signals, the concentration of HE4 could be calculated. Next, the performance of the established method was evaluated by the Clinical and Laboratory Standards Institute (CLSI) standards and National guidelines for Performance Testing for In Vitro Diagnostic Reagents. Results：The limit of blank (LoB), the limit of detection (LoD) and the limit of quantitation (LoQ) of the established method were 1.014 pmol/L, 5.252 pmol/L and 10.568 pmol/L, respectively. Under the conditions of AFP≤400 IU/mL, CEA≤1 777 μg/L, CA125≤3 500 U/mL and CA19-9≤3 500 U/mL, the detection of HE4 was not affected by their cross reactivity. The linearity range of the established method was from 20 pmol/L to 1 700 pmol/L, and there was no Hook effect at 100 000 pmol/L of HE4. The intraassay and interassay coefficients of variation were 1.5%~3.6% and 3.8%~6.4%, respectively, and the recovery rate was 98.36%~99.14%. Under the conditions of hemoglobin≤4.8 g/L, bilirubin≤1 077.5 μmol/L, chylomicrons≤6 000 turbidity, biotin≤50 μg/L and rheumatoid factor≤1 000 U/L, the detection of HE4 was not affected by their interference. After serum samples were stored at 37 ℃ for 7 days, the relative deviation of HE4 levels in serum was -6.63%~10.23%. In addition, there was a steady linear relationship with the equation Y=1.023X-12.280 between the established method (Y) and ELISA kit (X) from Fujirebio company (r=0.989 7, P<0.01). Conclusion：A chemiluminescence quantitative method for detecting HE4 based on micro0beads was successfully established, which had good performance and was conducive to the diagnosis of ovarian cancer.