ObjectiveTo establish Real-time PCR method for detection of woodchuck hepatitis virus (WHV) DNA. Methods13 pairs of primers were designed according to the coding sequence of woodchuck hepatitis virus core antigen (WHcAg) and woodchuck hepatitis virus surface antigen (WHsAg). The primers demonstrated high sensitivity and specificity were used for Real-time PCR detection of WHV DNA in woodchuck serum. Then, this method was applied in detection of WHV DNA in serum of woodchuck. ResultsThe selected primers pair (WHVSF1, WHVSR1) which based on 5′ end of WHsAg gene showed a detection limitation of 1×10^1copies/μL. The R² value of standard curve of viral copy number and the Real-time PCR Ct value was 0.997, and no mixed bands were observed during electrophoresis. ConclusionReal-time PCR method for detection of WHV DNA in serum of WHV infected woodchuck was established, which could be used in WHV model.