Herein, single-atom copper-modified Ti3C2Tx MXene nanosheets (CuSA-Ti3C2Tx) were introduced to boost electrochemiluminescence (ECL) of the luminol/O2 system, thereby establishing a sensing platform for ultrasensitive detection of enrofloxacin (ENR) related to residues of veterinary drugs in food. Unlike the conventional carbon-based materials, the oxygen vacancy-rich Ti3C2Tx substrate enabled the high-density immobilization of single-atom Cu sites on its catalytically active basal plane, which accelerated the oxygen reduction reaction at a low potential for generating abundant reactive oxide species (ROS) to significantly promote the ECL response of luminol. Notably, the innovative luminol/O2/CuSA-Ti3C2Tx ternary system could achieve a 15-fold enhancement in ECL intensity compared to that of the luminol/O2 binary system. Furthermore, less target triggered the entropy-driven chain displacement reaction to generate the massive ferrocene-labeled DNA nanowires for rapidly consuming ROS on the surface of CuSA-Ti3C2Tx, enhancing the detection sensitivity. As a result, the ECL biosensor realized the trace analysis of ENR with a low detection limit (LOD) of 0.86 fg/mL, far beyond the current rapid detection methods for antibiotics. Meanwhile, the proposed biosensor successfully was applied in the quantitative detection of antibiotics in animal foodstuff. The strategy proposed a reasonable approach to establish high-performance single-atom catalysts, which was prospective to monitor antibiotics in food quality control and environmental pollution monitoring.