We constructed a label-free and detector-free aptazyme-based riboswitch sensor for detecting the cofactor of the aptazyme. This riboswitch, which usually suppresses the gene expression with its anti-RBS sequence bound to the RBS of its own mRNA (OFF), activates the translation only when a cofactor is added to release the anti-RBS sequence from itself as a result of cofactor-induced self-cleavage by the aptazyme (ON). The rationally optimized one with beta-galactosidase as a reporter gene enabled us to detect the cofactor of the aptazyme visibly with high ON/OFF efficiency. (C) 2007 Elsevier Ltd. All rights reserved.