The 1.2 kb region of 5′ flanking sequence of bovine casein gene and human alpha lactalbumin gene was combined with the PSV vector which has SV40 promotor and β-galactosidase gene(LacZ gene). The expression vector αS1-LA-psv was successfully constructed. Bovine mammary epithelial cell was cultured. The biological characters including survival rate of cells inoculation, doubling time of cell population growth curve and morphology were detected after passaged and purified. And the cell line was identified by the tissue-special expression of cytokeratin 18. The normal cultured dairy bovine mammary epithelial cell line was set up. The cells could still have a good behavior of proliferation after passaged 20 times. The cells were transfected with the eukaryotic expression vector αS1-LA-psv and the activity of β-galactosidase was detected after transfected from 24 h to 120 h. Human alpha lactalbumin was detected at 72 h after transfected too. The production of human alpha lactalbumin was 0.64 g/L approximately. The results indicate that the bovine mammary epithelial cell line have the ability of expressing exogenous gene, the 5′ flanking sequence of bovine αS1 casein gene has the effect of regulating gene expression specifically and the vector αS1-LA-psv which was constructed can coexpress alpha lactalbumin and β-galactosidase.