A DNA vaccine against the hepatitis B virus (HBV), enhanced by IL‐2/IFN‐γ fusion protein expression from a plasmid construct and mediated by in vivo electroporation, was evaluated in a total of 39 HBeAg‐positive patients with chronic hepatitis B (CHB). The six of 39 patients with a serum alanine aminotransferase (ALT) value of 1–2 times upper limit of normal (ULN) were assigned to the open‐label arm (Group01) receiving vaccine monotherapy; the remaining 33 patients with an ALT of more than two times ULN were enroled to the randomized and controlled arm (Group02) receiving lamivudine (LAM) monotherapy (LAM+placebo) or combined therapy (LAM+DNA vaccine) in 1:2 ratio. In Group01, a significant elevation of HBV‐specific IFN‐γ‐secreting T‐cell counts in comparison with baseline was observed. In Group02, the proportion of patients with HBV DNA suppression was higher with LAM+DNA vaccine than with LAM monotherapy at each visit time point after the final injection of DNA vaccine at week 36, revealing a significant difference between the two groups (P = 0.03) at week 60. The incidence of dual‐site mutations of rtM204/I/S+rtL180M was significantly lower (P = 0.03) with an identified lower virological breakthrough (VBT) rate (P = 0.03) in patients receiving LAM+DNA vaccine than LAM monotherapy, accompanied with a significant higher positive T‐cell response rate in patients receiving LAM+DNA vaccine (P = 0.03). In conclusion, this study provides evidence that HBV DNA vaccination is safe and immunologically effective, and that the HBV‐specific T‐cell responses induced by DNA vaccination under LAM chemotherapy showed a correlation with the suppression of viral replication in patients with CHB.