Abstract: Objective To construct an expression vector for highly efficient expression of tissue-type plasminogen activator (t-PA) confined in the mammary gland of cow to develop a cow mammary gland bioreactor. Methods RT-Touch down-PCR was employed to amplify human tissue-type plasminogen activator (t-PA) cDNA, which was digested with the restriction enzymes and subsequently cloned into the vector pSP72 for constructing specific fusion gene only expressed in the mammary gland. The fusion gene was then transferred into the mouse zygote and the mammary gland tissue of mice and cows. Results t-PA was detected in the milks of mice and cows after the transgenic manipulation with microinjection and mammary gland injection of the fusion gene. Conclusions The vector we constructed can effectively induce t-PA expression in the mammary gland, which is not influenced by different transgenic methods. The expression level of t-PA, however, is significantly higher in the milk of cows than in the milk of mice, suggesting the species-specific difference in milk protein regulating system possibly is due to different factors and regulatory systems.