A series of plasmids derived from pNL201 with the promoter upstream region of alpha-amylase gene of B. licheniformis have been constructed. In the meantime, a series of plasmids derived from pAmy41 with full alpha-amylase gene have also been constructed and they have different promoter upstream region with the EcoRI-BclI fragment deleted or partial destroyed. The alpha-amylase activity of the engineering strains carrying alone or double plasmids has been determined. The statistical results show that the EcoRI-BclI fragment possesses negative regulation in the expression of alpha-amylase gene in B. subtilis.