In this paper, a novel and label-free fluorescence sensing strategy has been developed for tetracyclines (TCs) detection in food sample by using poly(thymine) (poly T)-templated copper nanoclusters (CuNCs) as fluorescent probes. Under optimized conditions, the label-free fluorescent sensor achieved highly sensitive and selective detection of TCs with a detection limit of 0.01 μmol/L, and with a detection limit of 48 μg/kg in real samples. Furthermore, the method was successfully applied to the detection of TCs in milk and chicken samples. The relative standard deviation was less than 4.01%, and the recovery rate was between 97.67% and 110.23%. The strategy was free of any fluorescence dye label or complex DNA sequence design, and all the processes were performed within 10 minutes at room temperature. Therefore, it could offer a simple, rapid, and label-free sensing platform for fluorescence detection of TCs in food.