Abstract: An indirect enzyme-linked immunosorbent assay(ELISA) method was developed to detect serum antibodies for duck Tembusu virus(DTV).ELISA plates were coated with the main antigenic determinant of the envelop proteins(E1protein).The optimized test conditions included:concentration of E1 coating on the ELISA plate of 1.548 mg·L-1,serum sample dilution at 1∶100,and HRP-labeled goat anti-duck IgG dilution at1∶6 000.The specific tests showed no cross-reactions on anti-sera against the avian influenza virus,Newcastle disease virus,duck hepatitis virus-I,duck hepatitis type 1virus with pancreatitis,pasteurellosis or riemerella anatipestifer,indicating a high specificity of the ELISA against DTV.The coefficients of variation of the intro-and inter-batch duplicability tests were below 10%.The sensitivity was 1∶3 200.Compared with the indirect ELISA coated with DTV,the coincidence rate of indirect ELISA was 95.1%.Out of the 237 laying duck serum samples collected from Fujian,194(i.e.,77.9% of the total)were found positive on the indirect ELISA.The newly developed indirect ELISA methodology appeared to have desirable specificity,repeatability,as well as sensitivity,and could potentially be applied for routine DTV detection.