Objective Duck hepatitis B virus (DHBV) infected animal model was used to evaluate the inactivation of DNA virus by methylene blue photodynamic method. Methods DHBV was purified by Ultra-centrifugation. Purified DHBV was sequentially diluted and added to human plasma or RBC components respectively, followed by inactivation with methylene blue photodynamic method. These samples were inactivated into one-day-old ducklings by intravenous injection. Sera from ducks were collected at different intervals and detected by dot hybridization with DHBV probe labeled with 32P for DHBV DNA, and confirmed by southern blot hybridization for DHBV DNA in duck livers. Results The infectivity of DHBV (ID50) in the blood components was calculated before and after the treatment with methylene blue photodynamic method. Before viral inactivation, the ID50 of DHBV mixed with human plasma for one-day-old duckling was 1033.3 copies, and the ID50 of DHBV mixed with human RBC in one-day-old duckling was 103.50 copies. After inactivation with methylene blue photodynamic, the ID50 of DHBV mixed with plasma in one-day-old duckling was 1010 copies, while the ID50 of DHBV-RBC in one-day-old duckling was 108.35 copies. Treatement of methylene blue photodynamic the viral titer of DHBV-plasma decreased 6 logs, and DHBV-RBC decreased 5 logs. Conclusion Methylene blue photodynamic can inactivate DNA virus(using DHBV as a marker) in human blood or RBC, and the inactivation effect is better for virus in the plasma than that in RBC. The DHBV infected ducks could be used as a model for evaluating the effect of DNA virus inactivation in vivo.