The key biological thiol, cysteine (Cys), which can participate in many physiological and pathological processes in the human body, has also been proved to have considerable effects on redox homeostasis and the regulation of cell activity in vivo. A large number of studies have shown that abnormal Cys concentration is inseparable from the occurrence of many diseases. Therefore, it is of considerable research value to develop a method to specifically detect Cys. In this study, a new ratiometric fluorescent probe of two-isophorone fluorophore, TIFC, was proposed to successfully detect intracellular Cys by ratiometric fluorescence signals and the results showed that TIFC has good selectivity and sensitivity. The results of biological imaging experiments also demonstrated that probe TIFC can exhibit a rapid ratiometric response to Cys in vivo and has low cytotoxicity, enabling high biocompatible fluorescence imaging in osteoblasts. Therefore, TIFC has the potential of rapid ratio-response to Cys for the preliminary diagnosis of related diseases.