Abstract: Objective：To investigate the relationship among HBV pre-S1 antigen(Pre-S1Ag)，HBV DNA and HBeAg, and to evaluate the clinical significance of Pre-S1Ag. Methods：Serum HBeAg and Pre-S1Ag were detected by ELISA in 178 patients with chronic Hepatitis B. HBV DNA was tested by quantitative fluorescence PCR technique. The positive concordance rate of HBV DNA (+)/Pre-S1Ag (+) and HBV DNA(+)/HBeAg(+) was compared. All patients were divided into three groups according to their HBV DNA levels: low duplication group, medium duplication group and high duplication group. The correlation between HBV DNA levels and Pre-S1Ag was analyzed. Results：In 178 patients with HBV DNA(+),Pre-S1Ag positive was 144 cases (80.9%) and Pre-S1Ag negative was 34 cases (19.1%). For HBeAg, 112 cases (62.9%) were positive and 66 cases (37.1%) were negative. The positive concordance rate of HBV DNA (+)/Pre-S1Ag(+) and HBV DNA(+)/ HBeAg (+) was 80.9% and 62.9%,respectively,there was statistically significant (P<0.05). In 112 cases with HBeAg (+)，the positive rate of Pre-S1Ag was 80.4%. In 66 HBeAg (-) cases, the positive rate of Pre-S1Ag was 82%. The positive rate of Pre-S1Ag in high duplication group was higher than in the medium’s (80%) and low’s (64%). The level of HBV DNA was significantly related to the positive rate of Pre-S1Ag(r = 0.293，P<0.01).Conclusion：There is a better correlation between the Pre-S1Ag and HBV DNA. In order to enhance the examination accuracy, Pre-S1Ag should be measured in chronic Hepatitis B patients with HBeAg (-) , if some hospitals are not able to detect HBV DNA.