One of the most pressing challenges in cell-free synthetic biology is to assemble well-controlled genetic circuits. However, no complex circuits have been reported in eukaryotic cell-free systems, unlike the case in bacterial ones, despite several unique advantages of the former. We here developed protein-responsive upregulating riboswitches (ON-riboswitches) that function in wheat germ extract to create multistep gene regulatory cascades. Although the initial two types of ON-riboswitches we first designed were less efficient than desired, we improved one of them by incorporating hybridization switches to successfully construct a pair of highly efficient, protein-responsive ON-riboswitches. Both upregulated expression up to 20-fold through self-cleavage by a hammerhead ribozyme (HHR) in response to the corresponding protein ligands expressed