Abstract: To simultaneously detect the presence of Newcastle disease virus (NDV), duck plague virus (DPV) and/or duck Tembusu virus (DTMUV) in ducks, 3 pairs of specific primers based on the conserved regions of the genomes of these viruses deposited in Gen Bank database were used for the development of a multiplex PCR assay method. The PCR reaction conditions were optimized, and specificity and sensitivity of the assay determined. The results showed that the new method could amplify the specific gene fragments of NDV at 510 bp, DPV at 400 bp, and DTMUV at 300 bp simultaneously without adulterations. The detection sensitivity of the assay was 1.02×10⁴ copies·μL^-1 on NDV, 3.50×10³ copies·μL^-1 on DPV, and 1.17×10⁴ copies·μL^-1 on DTMUV. The detection results were completely agreeable with those obtained by the conventional PCR method on 250 clinical samples. It appeared that the currently established methodology to simultaneously detect the 3 target viruses was rapid and convenient with high specificity and sensitivity.