Herein, a novel fluorometric-sensor with dual-emission system was constructed on the basis of polyvinylpyrrolidone (PVP) and 2-mercaptobenzothiazole (MBT) co-functionalized gold/copper nanoclusters (PVP/MBT-Au@CuNCs) by a facile and eco-friendly one-pot approach. The sensor exhibited ratiometric fluorescence emission (F590 nm/F422 nm) for visual and selective detection of S2− with a sensitive detection limit of 11.9 nM. Besides, fluorescence quenched sensing of S2− was chalked up by a quickly selectivity monitoring time of 30 s, owing to the strongly binding of Cu2S and Au2S by hard-soft-acid-base theory and the destruction of the aggregated structure of PVP/MBT-Au@CuNCs. Furthermore, the platform also provided the portable analysis for visual detection of S2− by capturing the change in fluorescence color with a single dual-emissive ratiometric paper strip. It is worth mentioning that the fluorescent gold-copper nanoclusters showed excellent application activities in the selective detection of S2− in Radix Codonopsis or Tremella samples and recognition of S2− in HeLa cells or macrophages by confocal microscopy fluorescent imaging. Overall, the sensing system paved a new avenue for effectively developing a convenient ratiometric fluorescent sensor platform for evaluating the safety of food with S2− pollution in environment and biological system.