Aptamer-based regulatory biosensors can dynamically regulate the expression of target genes in response to ligands and could be used in dynamic metabolic engineering for pathway optimization. However, the existing aptamer-ligand biosensors can only function with non-complementary DNA elements that cannot replicate in growing cells. Here, we construct an aptamer-based synthetic regulatory circuit that can dynamically upregulate and downregulate the expression of target genes in response to the ligand thrombin at transcriptional and translational levels, respectively, and further used this system to dynamically engineer the synthesis of 2'-fucosyllactose (2'-FL) in Bacillus subtilis. First, we demonstrated the binding of ligand molecule thrombin with the aptamer can induce the unwinding of fully complementary double-stranded DNA. Based on this finding, we constructed a bifunctional gene expression regulatory circuit using ligand thrombin-bound aptamers. The expression of the reporter gene ranged from 0.084- to 48.1-fold. Finally, by using the bifunctional regulatory circuit, we dynamically upregulated the expression of key genes fkp and futC and downregulated the expression of gene purR, resulting in the significant increase of 2'-FL titer from 24.7 to 674 mg/L. Compared with the other pathway-specific dynamic engineering systems, here the constructed aptamer-based regulatory circuit is independent of pathways, and can be generally used to fine-tune gene expression in other microbes.