Realizing the potential of cell-free systems will require development of ligand-sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand-sensitive transcriptional control in cell-free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single-stranded template, results in up to a 6-fold change in gene expression in a ligand concentration-dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell-free systems.