Abstract: Object: Establish rapid,sensitive and specific methods of detecting three double- strand DNA viruses,Channel catfish virus( CCV),Koi herpesvirus( KHV),Epizootic hematopoietic necrosis virus( EHNV),from fish samples.Method: Three pairs of specific primers and three Taqman probes were designed targeting for conserved sequence of DNA polymerase gene of three kinds of virus. The Triplex real- time PCR assay was established. The sensitivity and specificity of the method were evaluated.Results: The sensitivity of this method was 102copy·μL- 1for detecting DNA of three viruses. The assay was specified for three kinds of virus CCV,KHV and EHNV. In the experiment,non- interference between primers and probes was found.Conclusion: The Triplex real- time PCR assay was a stable and reliable method for rapid detection of three double- strand DNA viruses in aquatic animal products.