Objective: To explore the relationship between hepatitis B virus DNA (HBV-DNA) and HBV serum markers (HBV-M) as well as HBV pre-S1Ag. Methods: In all, 556 peripheral blood samples were prospectively collected from patients who are suffering from HBV at the Affiliated Hospital of Xuzhou Medical University between December 2016 and January 2018. HBV-M and pre-S1Ag were respectively detected with electrochemiluminescence and magnetic particles chemiluminescence method. The content of HBV-DNA was analyzed by fluorescence quantitative PCR (qRT-PCR). Results: The positive rates between HBV pre-S1Ag and HBV-DNA were significantly different among the different HBV-M mode group (P<0.05). In HBsAg, HBeAg, anti-HBc positive group (1, 3, 5 modes), the positive rate of HBV-DNA and HBV pre-S1Ag were 92.70% and 97.08% respectively. These quantitative indicators respectively reached 54.62% and 86.92% in HBsAg, HBeAb, anti-HBc positive group (1, 4, 5 modes). In the model of HBsAg (+)/HBeAg (+)/HBeAb (+)/anti-HBc (+) group, the positive rate of HBV-DNA and HBV pre-S1Ag were 95.83% and 100% respectively. In the model of HBsAg (+)/HBsAb/HBeAb (+)/anti-HBc (+), HBV-DNA detection rate was 41.67% and pre-S1Ag account for 79.17%. However, HBV-DNA and HBV pre-S1Ag were not detected in HBsAb, HBeAb, anti-HBc positive group (2, 4, 5 modes). Overall, the positive rate of HBV-DNA and HBV pre-S1Ag were respectively 92.74% and 94.97% in patients with HBeAg (+). Conclusion: The detection of HBV serum marker can help to diagnose HBV infection. HBV pre-S1Ag is a better index, which can determine the infection, reproduction and activity of HBV, however, it still can’t substitute HBV-DNA. HBV pre-S1Ag, a good parameter in judgment the infection, replication and activity of HBV, can’t replace the HBV-DNA quantification. The combination of the HBV-DNA quantification and the HBV serum markers detection can improve the diagnostic efficiency of hepatitis B.