Abstract: The aim of this study was to construct mammary gland specific bicistronic expression vector based on FMDV 2A, and verify the expression of sequences of EGFP and human IL2 in mammary epithelial cells. We utilized selfcleaving peptide 2A from footandmouth disease virus (FMDV 2A) connecting hIL2 and EGFP sequence, and cloned into goat βcasein regulatory sequence, then we got mammary gland specific bicistronic expression vector pFIENβ, after transient transfection into goat mammary epithelial cells by LipofectamineTM 2000, we used RTPCR and Western blot to detect the expression of hIL2 and EGFP. Restriction analysis suggested that the recombinant plasmid pFIENβ is correct, then we found that the cells which were transfected with pFIENβ and pEGFPC1 expressed EGFP by observing with fluorescent microscope, and there is no green flourescence in which were untransfected, and we amplificating hIL2 and EGFP sequence in the cells transfected with pFIENβ, and amplificating EGFP sequence only in the cells transfected with pEGFPC1; After detection by Western blot, the cells transfected with pFIENβ expressed both hIL2 and EGFP. The results showed that goat βcasein regulatory sequence can trigger hIL2 and EGFP expression in mammary epithelial cells, and FMDV 2A facilitate expresson of hIL2 and EGFP independently.