This study aimed to develop a novel and practical fluorescent method for GSH detection in complex biological samples. To this end, a series of coumarin-based fluorescent probes was designed and synthesized using various aliphatic halogens as the sensing group. By using a new evaluation method of GSH/Cys/Hcy coexisting conditions, the probe with chloropropionate (CBF3) showed a high selectivity, excellent sensitivity, good stability for GSH detection. The reaction mechanism is proposed as nucleophilic substitution/cyclization and intramolecular charge transfer (ICT), which was confirmed by LC-MS and NMR analysis, as well as density functional theory calculations. In addition, CBF3 was demonstrated to be competent not only for the quantitative detection of GSH in real serum samples, but also for sensing GSH changes in different oxidative stress models in living cells and nematodes. This study showed a practical strategy for constructing GSH-specific fluorescent probes, and provided a sensitive tool for real-time sensing of GSH in real biological samples. The findings would greatly facilitate further investigations on GSH-associated clinical diagnosis and biomedical studies.