In this study, a coumarin-based probe (probe 1) bearing a maleimide group was used to rapidly and selectively detect thiols. After inducing thiols, this probe presented a significant fluorescence enhancement (≥22-fold), whereas other amino acids that had no sulfhydryl group just generated slight changes. The detection limits were determined to be as low as 7.41 nM for Cys, 6.06 nM for GSH, and 7.73 nM for Hcy based on S/N = 3. Furthermore, this probe possessed excellent water solubility and successfully detected thiols in PBS (pH 7.4, 99.5%). More importantly, the practical application of probe 1 for the selective detection of thiols was successfully demonstrated in living T24 cells.