Translational Initial Region (TIR) is the threshold of an intracellular translation process, and tiny alterations in this region are reported to intensely influence the downstream expression. Such property provides a potential utilization in extracellular production of recombinant enzyme. As an esterase, cutinase is an essential catalyst in the process of textile scouring, and has a potential application in food and chemical industry. In the present study, a bacterial cutinase (Tfu_0883) from Thermobifida fusca was expressed in Escherichia coli with pelB as its signal peptide using trc as its promoter. A subsequent TIR degeneracy mutagenesis was then carried out in the initial sequence of pelB. A fast screening method for these mutants was developed and a series of strains with different expression strengths were accordingly obtained. Among these mutants, a high cutinase production level of 38.0 U/ml was achieved, which is three times that of the control group. This study explored the potential utilization of TIR degeneracy mutagenesis in the production of industrial enzymes.