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Rapid and accurate detection of Escherichia coli (E. coli) is critical for maintaining water quality, and protecting aquatic ecosystems and public health. This research focuses on the development of a Förster resonance energy transfer (FRET)-based "turn-on" fluorescent nanosensor for real time, sensitive detection of E. coli. Copper nanoclusters-encapsulated metal organic frameworks (CuNCs@ZIF-8) were sythesized as a fluorescent donor with excellent luminescence properties. Further, MnO 2 nanospheres were synthesized as a receptor with good adsorption and quenching abilities. This novel nanoconjugate (CuNCs@ZIF-8@ MnO 2 ) was employed for the construction of a sensitive, accurate, and rapid sensing platform against E. coli in water on the basis of p-benzoquinone/hydroquinone (p-BQ/HQ) redox pair formation. Fluorescence is quenched by energy transfer when MnO 2 nanospheres are added to CuNCs@ZIF-8. Upon contact with E. coli, NADH-quinone reductase converts p-BQ to HQ, which reduces MnO 2 to Mn 2+ , releasing the nanospheres and restoring fluorescence in the composite. Based on this FRET ON-OFF-ON fluorescent probe, E. coli can be detected across a broad concentration range (5 × 10 1 to 5 × 10 5  CFU/mL), with a detection limit as low as 8 CFU/mL within 50 min. The sensor's practicality was verified through the investigation of E. coli in real water samples, with recoveries in the range 94.3 to 106.5%. This approach offers an efficient method for on-site detection and quantification of E. coli in both environment and food safety domains., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)