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Bacterial infections are a global healthcare problem, resulting in serious clinical morbidities and mortality. Real-time monitoring of live bacteria by fluorescent imaging technology has potential in diagnosis of bacterial infections, elucidating antimicrobial agents' mode of action, assessing drug toxicity, and examining bacterial antimicrobial resistance. In this work, a naphthalimide-derived fluorescent probe ZTRS-BP was developed for wash-free Gram-positive bacteria imaging. The probe aggregated in aqueous solutions and exhibited aggregation-caused fluorescence quenching (ACQ). The interaction with Grampositive bacteria cell walls would selectively disaggregate the probe and the liberated probes were dispersed on the outside of the bacteria cell walls to achieve surface fluorescence imaging. There were no such interactions with Gram-negative bacteria, which indicates that selective binding and imaging of Gram-positive bacteria was achieved. The binding of zinc ions by ZTRS-BP can enhance the fluorescent signals on the bacterial surface by inhibiting the process of photoinduced electron transfer. ZTRS-BP-Zn(II) complex was an excellent dye to discriminate mixed Gram-positive and Gram-negative bacteria. Also, live and dead bacteria can be differentially imaged by ZTRS-BP-Zn(II). Furthermore, ZTRS-BP-Zn(II) was used for real-time monitoring bacteria viability such as B. cereus treated with antibiotic vancomycin.