A new ELISA inhibition test for measuring serum Pre-S2Ab was developed to avoid the "double positive" phenomenon. One hundred and twenty samples with Pre-S2Ab negative were detected for Pre-S2Ab, the critical range for Pre-S2Ab in normal population was confirmed: a positive result was decided while the inhibition ration was over 50%, a negative result was decided while the inhibition ratio was less than 34%, and a dubious result was decided while the ratio was over 34% but less then 50%. At the same time, serum samples from sixty patients with chronic hepatitis B were detected for Pre-S2Ag and Pre-S2Ab, it showed that (1) the positive ratio of HBV DNA in patients with Pre-S2Ag positive was much higher than in patients with Pre-S2Ag negative (P < 0.01); (2) to the 46 patients with HBV DNA positive, Pre-S2Ag had a better relationship with HBV DNA than the HBeAg/Ab did (P < 0.01). Furthermore, to patients with acute hepatitis B, the presence of Pre-S2Ab was closely related to the prognosis of the disease, and the Pre-S2Ab assay could also be used to evaluate the preventive effect of HB vaccine containing Pre-S2Ag.