The aim of this study is to investigate the effect of hyperin on the cccDNA of duck hepatitis B  virus and its immunological regulation.  Duck hepatitis B virus (DHBV) infection model and normal mouse spleen lymphocyte were used to evaluate the anti-HBV and immunoregulation effects.  The DHBV-DNA of serum was detected at different time points by using serum DOT-BLOT hybridization.  Polymerase chain reaction (PCR) was used for the determination of nuclear covalent closed circular DNA (cccDNA).  Cytokine secretion was determined by ELISA method.  DHBV-DNA were inhibited by hyperin (25 or 50 mg·kg⁻¹), while cccDNA of liver could be eliminated efficiently by hyperin (25 or 50 mg·kg⁻¹, P < 0.05, P < 0.01).  The T helper 1     effector cytokine was markedly enhanced by hyperin (25 or 50 μg·mL⁻¹, P < 0.01).  In conclusion, hyperin has anti-HBV activity via multiple targets and pathways, and cccDNA may be one of the important targets.