Objective To construct recombinant eukaryotic expression vectors of Beagles ERβ₁₂₉₃, and identify the expression and localization of ERβ₁₂₉₃ in the human embryonic renal HEK293T cells by Western blotting and indirect immunofluorecent assay.Methods The ERβ₁₂₉₃ gene was amplified by PCR using pEGFP-N1-ERβ₁₂₉₃ as a template. The recombinant eukaryotic expression vector of pcDNA3.1-Myc-ERβ₁₂₉₃ was constructed, and transfected it into HEK293T cells. The expression of pcDNA3.1-Myc-ERβ₁₂₉₃ was detected by Western blot, and its localization was detected by indirect immunofluorecence assay. Results The expression of pcDNA3.1-Myc-ERβ₁₂₉₃ was constructed, and successfully identified in the HEK293T cells using Western blotting, and detected only in the cytoplasm using IF by laser scanning confocal microscopy. Conclusions In the previous experiment, the coding sequence of Beagles ERβ₁₅₉₃ splice variants has been obtained, which is lack of exon 4, and its ligand binding ability is reduced or disappeared, so that the intracellular localization of ERβ₁₂₉₃ codeing protein is changed.