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 ultrasonication method. This moiety was confirmed through structural elucidation by FTIR, and, NMR spectroscopy techniques. Further, the photophysical properties of the fluorescent moiety were tested using UV- Visible and Emission spectroscopy techniques. In this case, the moiety was tagged with antibody of Enterobacter cloacae through 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-Hydroxy succinimide (EDC/NHS) coupling and applied as sensing element for the detection of Enterobacter cloacae (E.cloacae) by using UV- Visible and Emission spectroscopy techniques. Developed fluorescent sensor has detected the E. cloacae via FRET mechanism. Under optimized conditions, ACC-anti-E.cloacae has detected E.cloacae in the linear range from 101 to 1010 CFU/ mL with the limit of detection (LOD) of 10.55 CFU/mL. The developed sensor has been applied for determination of E.cloacae in food samples like orange, pomegranate, milk, rice, tomato, potato and onion. Keywords: Enterobacter cloacae, Immunoassay, Chromene, Ultrasonication, Food samples, ELISA