Corn steep liquor was used as the sole carbon and nitrogen source for recombinant E. coli culture medium. Based on the single factor optimization, three factors of corn steep liquor, MgSO₄ and trace elements were chosen to design the response surface of corn steep liquor medium components. With Box-Behnhen experimental design and response surface analysis, the optimum components of corn steep liquor medium were determined. The results showed that the optimum medium components were 26 g/L for corn steep liquor, 2.1 g/L for MgSO₄ and 7 mL/L for trace elements. Under these conditions, the activity of trehalose synthase produced by recombinant E. coli reached 120.5 U / mL. The experiment proved that this medium is feasible, in which corn steep liquor was the sole carbon and nitrogen source of trehalose synthase.