Cell-based sensors are useful for many synthetic biology applications, including regulatory circuits, metabolic engineering, and diagnostics. While considerable research efforts have been made toward recognizing new target ligands and increasing sensitivity, the analysis and optimization of turn-on kinetics is often neglected. For example, to our knowledge there has been no systematic study that compared the performance of a riboswitch-based biosensor versus reporter for the same ligand. In this study, we show the development of RNA-based fluorescent (RBF) biosensors for guanidine, a common chaotropic agent that is a precursor to both fertilizer and explosive compounds. Guanidine is cell permeable and nontoxic to E. coli at millimolar concentrations, which in contrast to prior studies enabled direct activation of the riboswitch-based biosensor and corresponding reporter with ligand addition to cells. Our results reveal that the biosensors activate fluorescence in the cell within 4 min of guanidine treatment, which is at least 15 times faster than a reporter derived from the same riboswitch, and this rapid sensing activity is maintained for up to 1.6 weeks. Together, this study describes the design of two new biosensor topologies and showcases the advantages of RBF biosensors for monitoring dynamic processes in cell biology, biotechnology, and synthetic biology.