Objective To explore the influence of enzyme-linked immunosorbent assay method of non-equilibrium on the detection of e antibody and core antibody in hepatitis B. Methods 44 cases of anti-HBe positive and 57 cases of anti-HBc positive specimens determined by electrochemical luminescence immunoassay were analyzed by enzyme linked immunosorbent again. The enzyme marker were added into reaction holes at 0 min, 30 min after samples add respectively, the remaining steps were followed in strict accordance with the manual operation. Results The miss positive detection rate of anti-HBe and anti-HBc analyzed by equilibrium method were 52.3% and 40.4% respectively. The miss positive detection rate of anti-HBe and anti-HBc analyzed by non equilibrium method were 34.1% and 24.6% respectively. Conclusion It would be better to prolong the adding time of enzyme in the detection of anti-HBe and anti-HBc by enzyme-linked immunosorbent assay in order to increase the detection sensitivity.