Objective To construct and identify retroviral-mediated short hairpin RNA(shRNA) expression vectors of ERβ419, and explore ERβ419 unknown biological function in beagles in future. Methods To screen out the most effective gene silencing sequence of beagle ERβ419 mRNA using qRT-PCR and Western Blot assays, imitate beagle estrogen target cells. Results qRT-PCR results showed,ERβ419-shRNA1(P <0.01)and ERβ419-shRNA3(P<0.01)differed significantly, Western Blot result as same as qRT-PCR,ERβ419-shRNA3 is the best choice. Conclusion Beagles ERβ419-shRNA3 retrain most effectively target gene repression. It is applied to explore ERβ419 unknown biological function in beagles reproductive system, and to prevent and treat beagles reproductive function diseases.