Fluorescent biosensors are the molecules that change their fluorescence properties upon reaction with a substance of interest. They are widely used in biological/pharmacological research because they visualize the dynamic behavior of targets such as Ca²⁺ with high sensitivity in living cells/tissues. To date, two types of fluorescence sensors have been well studied: small molecule sensors based on synthetic fluorescent dyes and genetically encoded sensors based on (green) fluorescent proteins. Recently, attempts have been also made to develop the third class of sensors called “chemi-genetic” sensors, which are composed of both proteins and small molecules. However, most chemi-genetic sensors so far are based on the uniform molecular design in which an environmentally sensitive synthetic fluorescent dye is covalently bound to a HaloTag protein that is genetically fused with a target-binding protein. In addition, protein engineering of these sensors has not been sufficiently pursued. To address these issues, we have first demonstrated a completely new design of chemi-genetic Ca²⁺ and Na⁺ sensors consisting of a small molecule chelator and a fluorescent protein. In a separate project, we also developed a high-performance chemi-genetic K⁺ indicator through extensive protein engineering.