Riboswitches are natural RNA-based gene regulatory elements found within messenger RNA (mRNA), predominantly in bacteria, as well as in certain archaea, fungi, and plants. Natural riboswitches, with their inherent cis-regulatory, protein-independent, small-molecule-responsive regulation mechanisms, have prompted the creation of synthetic counterparts called synthetic riboswitches. Synthetic riboswitches comprise three essential domains: a sensor, a transmitter, and an actuator. Together, these integrated domains precisely control gene expression at transcriptional, post-transcriptional, and translational levels. The modular nature of these components enables researchers to design synthetic riboswitches that can sense specific non-endogenous molecules and precisely control desired gene expression spatiotemporally, making them a promising tool for biomedical applications. In this chapter, the authors reviewed approaches employed for designing individual riboswitch components, as well as the creation of single-input and multiple-input riboswitches (genetic circuits) and their new applications in biosensing, metabolic engineering, and therapeutic gene regulation.