The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds.