Objective To develop Enzyme linked Immunoassay for Hepassocin measurement. Methods Enzyme linked Immunoassay (ELISA) for Hepassocin in serum was developed using a double antibody sandwich method, in which one strain was coated on the microplate and another strain was labeled with horseradish peroxidase (HRP). TMB color was used as Chromogenic system. Results The Hepassocin detection range was from 5 to 120ng/mL. The correlation coefficient of the standard curve was more than 0.995, and the lowest detection level was 10ng/mL. The average recovery rate was between 93.9% and 103.1%. The intra assay CV was between 9.7% and 7.8%, and the Hepassocin normal concentration in serum of healthy men was less than 20ng/mL. Conclusion The detection results with 1000 serum samples by ELISA developed in this study showed good correlation with clinical results. The ELISA had same clinical values as the control kits.