The aim of this study was to investigate the diagnostic value of the serum markers HBsAg and HBeAg and PreS1 protein (PreS1-Ag) in quantifying the levels of hepatitis B virus (HBV) DNA in patients with chronic hepatitis B (CHB). One thousand CHB patients were recruited from Beijing You'an Hospital between June and December 2012. Serum HBsAg and HBeAg levels were detected by electrochemiluminescence immunoassay. Enzyme-linked immunosorbent assay and fluorescence quantitative PCR were used to determine the level of PreS1-Ag and HBV DNA, respectively. We observed a low correlation between HBsAg and HBV DNA (r = 0.172, P < 0.001) expression; however, the correlation coefficient increased gradually with the increase in HBV DNA levels, and was more significant when HBV DNA log10 > 7 (r = 0.597, P < 0.001). Additionally, HBsAg and HBV DNA showed a significant positive correlation in the HBeAg+ group (r = 0.321, P < 0.001), whereas no correlation was observed in the HBeAg- group (r = -0.016, P = 0.825). HBV DNA expression was correlated with HBeAg (χ2 = 83.07, P < 0.001) and PreS1-Ag (χ2 = 36.01, P < 0.001). HBV DNA-positive rate was higher in HBeAg/PreS1-Ag++ patients (72.26%) than that in the single-positive groups (P < 0.001). Therefore, serum HBsAg is not a good marker for the prediction of HBV replication, and co-detection of HBeAg and PreS1-Ag, which can better predict HBV DNA replication, can be used as a reliable method for the clinical diagnosis and treatment of CHB.