Objective:To establish a sandwich ELISA for quantitative measurement of ProMBP. Methods:Anti-ProMBP monoclonal antibodies(mAbs) prepared by our laboratory were purified by Protein A affinity chromatography and analyzed by SDS-PAGE and Western blotting to test their characteristics. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method, and antibody pairs were performed using anti-ProMBP mAb as coating antibody and HRP labeled anti-ProMBP mAb as labeled antibody,which optimal concentrations were defined by titration. Standard curve was performed using purified PAPP-A/ProMBP tetramer and was judged by sensitivity,reproducibility and recovery rate. The ProMBP levels of sera were measured with this assay. Results:The optimal paired antibodies were anti-ProMBP mAb 4C6E5B9 and HRP labeled anti-ProMBP mAb 9G4A6G10 which optimal concentrations were 2.5 μg/ml and 1:3 000,respectively. The sensitivity of this assay was 2.0 ng/ml. The coefficients of variation were 4.6% to 6.2% within assay and 4.6% to 10.5% between assays. The recovery rate was 92% to 113%.The ProMBP levels of serum were(26.37 ± 2.90)ng/ml,(357.71 ± 33.60)ng/ml and(1088.77 ± 58.13)ng/ml, which were collected from normal non-pregnant women,9 to 11 weeks gestation and 15 to 23 weeks gestation respectively. Conclusion:A sandwich ELISA assay for detecting ProMBP was obtained successfully.