Biothiols, comprising cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a pivotal role in maintaining the delicate redox balance essential for life processes, intricately intertwined with the well-being of living organisms. Deviations in their concentrations can precipitate a range of diseases. Herein, we presented a novel fluorescent probe, DIcou-DNBS, derived from coumarin, which possessed red emission and a substantial Stokes shift (122 nm), tailored for biothiol detection. DIcou-DNBS demonstrated rapid-responsiveness (6 min) towards Cys, Hcy and GSH with detection limits of 0.015 µM, 0.032 µM and 0.059 µM, respectively. Furthermore, its excellent biocompatibility enabled successful applications in biothiol fluorescence imaging of living cells and zebrafish. Beyond biothiol sensing, DIcou-DNBS leveraged its unique property to indirectly detect β-lactamases, making it a valuable tool in inhibitor screening and susceptibility testing of both sensitive (Staphylococcus aureus ATCC 29213) and resistant (Enterobacter cloacae ATCC 13047) bacterial strains. This assay system anticipates to furnish a potent instrument for visualization studies across biological and medical domains.