The effective identification and quantitative determination of is a major public health concern. Here, an innovative strategy that combines a bacteria-imprinted polydimethylsiloxane film for bacterial recognition and fluorescence resonance energy transfer platform for turn-on fluorescence sensing is demonstrated. The bacteria-imprinted polydimethylsiloxane film was facilely fabricated to generate corresponding specific sites on the polydimethylsiloxane surface via stamp imprinting using as template followed by modification with 1H,1H,2H,2H-perfluorooctyltriethoxysilane. The fluorescence resonance energy transfer platform was developed through electrostatic interaction between citrate-functional copper clusters and dopamine-stabilized gold nanoparticles. When the are present, the 1H,1H,2H,2H-perfluorooctyltriethoxysilane-modified bacteria-imprinted polydimethylsiloxane film can precisely capture the target; subsequently, the negatively charged bacteria compete with citrate-functional copper clusters and bind to dopamine-stabilized gold nanoparticles, leading to the fluorescence recovery of citrate-functional copper clusters. The entire detection process was achieved within 135 min, showing a wide linear calibration response from 10 to 1 × 10 cfu mL with a low detection limit of 11.12 cfu mL. Furthermore, the recoveries from spiked samples were from 97.7 to 101.90% with relative standard derivations lower than 10%. The established label-free assay of measuring is rapid, sensitive, specific, and efficient.