Abstract: 【Objective】 To construct bovine interleukin-17 (bIL-17) gene eukaryotic expression vector and detect its expression in bovine mammarily epithelial cells which were primarily cultured in vitro. 【Method】 Bovine interleukin-17 gene(bIL-17) was amplified from the spleen tissue of cow by RT-PCR, which was inserted into pMD19-T vector and then sequenced. The bIL-17 was inserted into an expression vector pEGFP-N3 carrying the enhanced green fluorescent protein to generate a new plasmid pEGFP-N3-bIL-17. The pEGFP-N3-bIL-17 eukaryotic expression vector was transfected into bovine primary mammary epithelial cells with Lipofectamine^TM2000 liposome. After the transfection, the green fluorescent protein was observed under fluorescence microscopy, and bIL-17 transcription was examined by RT-PCR and bIL-17 protein expression in cells detected by ELISA methods. 【Result and conclusion】 The recombinant vector pEGFP-N3-bIL-17 with a green fluorescence and neomycin resistance was constructed and bIL-17 protein could be normally expressed in mammary epithelial cells.