Live cell-based biosensors have emerged as a useful tool for biotechnology and chemical biology. Here, we designed genetically encoded sensor cells using intein-mediated protein engineering approach. Intein-mediated protein engineering has been utilized for various biological applications, including biosensing and bioimaging. In the presentation, we discuss the generation of genetically encoded biosensors that can report the presence of biologically active molecules via fluorescence-translocation based on split intein-mediated conditional protein trans-splicing (PTS) and conditional protein trans-cleavage (PTC) reactions. Our approach fully valued the bond-making and bond-breaking features of intein-mediated reactions in sensor construction and thus eliminated the interference of false-positive signals resulting from the mere binding of fragmented reporters. These live cell-based sensors were able to detect biologically active signaling molecules, such as Ca²⁺ and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca²⁺ stimuli and the cortisol agonist Dexamethasone. These live cell-based sensing systems were also utilized for the screening of hormone-like natural products. This approach holds large potential for applications such as drug screening and toxicology studies, which require functional information about targets.