His-tagged Chinese hamster dihydrofolate reductase (DHFR) expression in pET vector system has been reported very low. A newly modified pET protein expression vector, pET-DB, was utilized to overexpress of it in soluble form. The amount of DHFR reaches to 46% of the total protein in E.coli cells. This His-tagged DHFR could be purified routinely by Ni-NTA agarose resin and the His-tag could be removed by thrombin easily. This engineered DHFR has the same enzyme activity as the enzyme without His-tag obtained by iso-electrophoresis.