Duck enteritis virus (DEV) causes substantial losses on duck farms; however, its molecular biology is poorly understood. Here, an open reading frame of a US3-like gene of DEV was identified from a DEV genomic library. Its existence was confirmed by cloning from DEV-infected duck embryo fibroblasts (DEFs) and DNA sequencing. The US3-like gene was then subcloned into a prokaryotic protein expression vector and expressed as a six-histidine–tagged fusion protein in Escherichia coli. The protein was purified and inoculated into rabbits for antiserum production. A primary antibody specific to the gene was successfully generated and used to detect the US3-like protein in DEV-infected duck cells. In vivo expression of the US3-like protein in DEV-infected DEFs was demonstrated with indirect immunofluorescence assay and regular fluorescence microscopy, whereas uninfected DEFs did not show any specific fluorescent staining. Furthermore, indirect immunofluorescence assay and confocal microscopy were used to study the time course and subcellular localization of the protein expression. The protein was found to be expressed as early as 2 hr postinfection, and its expression was increased by time at 4, 8, 12, and 24 hr postinfection. The protein was found to be localized mostly around the perinuclear area and in the cytosol, and also in the nucleus at later time points. In addition, a US3 protein phylogenetic tree was constructed and showed that the evolutionary relationship of DEV is close to the genus Mardivirus. In short, the DEV US3-like gene and its in vivo protein expression were found for the first time, and DEV classification and the gene's functions were suggested.