According to the genome sequences of duck hepatitis A virus type-1(DHAV-1), one pair of specific primers was designed to amplify the 3D gene using RT-PCR. The amplified fragment was cloned into prokaryotic expression vector pET-32a and the recombinant plasmid was transformed into BL21(DE3) E.coli. Recombinant proteins were successfully expressed following IPTG induction. SDS-PAGE showed that the recombinant expression protein was about 68 kD. Western blot assay revealed that the desired proteins could be recognized by the monoclonal antibody against histidine-tagged proteins. The antiserum against the recombinant protein was produced by the immunized ICR mouse with recombinant proteins. Western blot results showed that the antiserum could react specifically with DHAV-1 allantoic fluid. These results showed that the 3D gene was successfully expressed in E.coli and the antiserum could be used for detection of the 3D protein, which would lay a foundation for function study of the 3D protein.