Duck Tembusu virus (DTMUV), a mosquito-borne flavivirus, has been identified as a causative agent of an emerging disease in ducks. Since its first report in 2010, several clusters of DTMUV have increasingly been identified and caused outbreaks in many Asian countries. This highlights the need for improved and novel broad detection assays in order to detect all circulating clusters of DTMUV. In this study, a universal one-step reverse-transcription PCR (RT-PCR) assay targeting a highly conserved region of the NS5 gene was developed and validated for broad detection of all DTMUV clusters. The newly developed universal RT-PCR assay could specifically detect all clusters of DTMUV without cross-reactions with common duck viruses and other related flaviviruses. The assay was able to detect DTMUV as low as a 0.001 50% embryo lethal dose/milliliter. The performance of the assay was evaluated by using experimental and field clinical samples. The assay could successfully detect DTMUV in all experimentally DTMUV-infected samples and gave a higher DTMUV detection rate (36%) than the previously reported envelope-specific RT-PCR assay (30%) in field clinical samples. All the positive samples were confirmed DTMUV-positive by DNA sequencing. In conclusion, the newly developed universal RT-PCR assay exhibited high accuracy, specificity, and sensitivity in broad DTMUV detection, thus providing an improved screening assay for routine detection and epidemiologic surveillance of DTMUV.