Duck Tembusu virus (DTMUV) was first isolated in Northern China in 2010, and is an emerging viral pathogen that is now rapidly spreading to most Chinese provinces causing a severe disease and economic losses for the duck breeding industry. The virus can affect ducks, chickens and geese, but currently there are no commercial ELISA methods for detecting antibodies against DTMUV from different species, or for assessing antibody titers in infected birds. In the present study, a solid-phase competition ELISA for this purpose was developed. The sensitivity and specificity of this test were 97.6% and 100%, respectively, when the percent inhibition (PI) was set at 15% using a serum dilution of 1:16. No cross-reaction with the sera against other avian viral pathogens was found in the study and the ELISA results generated for duck and chicken sera from the field were totally consistent with those of the classical virus neutralization assay. Therefore, this solid-phase competition ELISA represents a sensitive, specific and quantitative tool for the serological detection of Tembusu virus in different birds to facilitate the prevention and control of this disease.