Abstract: A riboregulator called toehold switch has been designed. In this riboregulator, the start codon (AUG) and ribosome binding site (RBS) are located on the loop of the hairpin structure, resulting in the stem structure with a completely complementary double-stranded RNA. Trigger RNA can open the hairpin through strand displacement reaction, and activate the downstream expression of green fluorescence protein (GFP), leading to a certain increasing of fluorescence signal, eventually realizing the regulation of E. coli gene expression. The effects of stem length on the expression of GFP were investigated. Results showed that the hairpin structure of toehold switch could effectively inhibit the expression of green fluorescent protein when the length of stem was more than 8 bp. Further co-expression experiments showed that trigger RNA could open the structure of hairpin RNA, resulting in a certain increasing of expression of GFP, and regulate the gene expression of E. coli. The E. coli gene expression system regulated by toehold switch is scalable and can be applied to the regulation of multi-gene expression, which has potential application value in the diagnosis and treatment of gene diseases.