Diverse chemical species such as ions and molecules exist within living cells and organisms undergoing dynamic changes in their local environment by a web of continuously interacting reactions. Reaction‐based fluorescent probes with a highly sought reversible feature can provide a real‐time monitor of the concentration dynamics (increases and decreases) of such chemical species, thus ideally suited to understand the physiological function, and pathogenic mechanisms of corresponding bio‐species in the regulation of cellular function and disease progression. This review summarizes the current methods for constructing reversible reaction‐based fluorescent probes. The sensing mechanisms and biological applications of these probes are also discussed. The representative examples reported recently are categorized according to the type of reversible chemical reactions utilized: nucleophilic additions (Michael additions, chromophore reactions), nucleophilic addition‐condensation reactions, and redox reactions. Finally, we present the potential challenges and suggestions for developing probes based on dynamic and reversible covalent bond formation reactions.