A recombinant UL55 protein (pUL55) of duck enteritis virus (DEV), produced in Escherichia coli, was tested for diagnostic applicability in an indirect enzyme-linked immunosorbent assay (I-ELISA) as a coating antigen. Serum dilutions of 1∶6400 (0.025 µg) are the maximum detection limits of the pUL55-ELISA, according to the determined cut-off value of 0.330. Antigenic cross-reactivity investigation in heterologous sera of ducks failed to provide evidence that other viruses of ducks could hamper the serodiagnosis of DEV, and the inhibition assay revealed that the specific binding of antigen and antibody can be inhibited by pUL55, both of which demonstrated a good specificity of the established pUL55-ELISA. This assay was further validated by comparison with a commercial I-ELISA based on DEV (DEV-ELISA) and a neutralization test (NT). The results suggested that the sensitivity of pUL55-ELISA was almost as good as DEV-ELISA but was much higher than NT. The established pUL55-ELISA is a rapid, simple, sensitive, specific, and inexpensive serodiagnosis for detecting antibodies against DEV and has a potential to complement the traditional assays for serodiagnosis of DEV; it can be used as a diagnosis alternative candidate for serologic surveillance of DEV infection.