For the first time, a novel fluorescent moiety, 2-amino-4-(7-formyl-1,8-dihydropyren-2-yl)-7-hydroxy-4H-chromene-3-carbonitrile (ACC), was synthesized by an ultrasonication method. The synthesis of this moiety was confirmed via structural elucidation using FTIR and NMR spectroscopy techniques. Further, photophysical properties of the fluorescent moiety were tested using UV-visible and emission spectroscopy techniques. In this case, the moiety was tagged with an antibody of Enterobacter cloacae via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC/NHS) coupling and applied as a sensing element for the detection of Enterobacter cloacae (E. cloacae) by UV-visible and emission spectroscopy techniques. The developed fluorescent sensor detected E. cloacae via a FRET mechanism. Under optimized conditions, ACC-anti-E. cloacae detected E. cloacae in the linear range from 101 to 1010 CFU mL−1 with a limit of detection (LOD) of 10.55 CFU mL−1. The developed sensor was applied for the detection of E. cloacae in food samples such as orange, pomegranate, milk, rice, tomato, potato and onion. © 2024 Elsevier B.V., All rights reserved.