To determine the distribution of duck plague virus (DPV) gE protein in paraformaldehyde-fixed, paraffin-embedded tissues of experimentally DPV-infected ducks, an indirect immunoperoxidase assay was established to detect glycoprotein E (gE) protein for the first time. The rabbit anti-His-gE serum, raised against the recombinant His-gE fusion protein expressed in Escherichia coli BL21 (DE3), was prepared and purified. Western blotting and indirect immunofluorescence analysis showed that the anti-His-gE serum had a high level of reactivity and specificity and could be used as the first antibody for further experiments to study the distribution of DPV gE protein in DPV-infected tissues. A number of DPV gE proteins were distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, duodenum, jejunum, ileum, and kidney of DPV-infected ducks and a few DPV gE were distributed in the Harders glands, myocardium, cerebrum, and lung, whereas the gE was not seen in the skin, muscle, and pancreas. Moreover, DPV gE was expressed abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of gE expression and distribution in infected ducks but also for understanding the pathogenesis of DPV.