<p>An innovative electrochemical biosensor is proposed for the sensitive detection of ofloxacin (OFL) by integrating exonuclease I (Exo I)–assisted recognition-transduction strategy into the MXene/AuNP-enhanced electrochemical platform. Through ingenious design, the presence of OFL triggers the competitive displacement reaction and enables Exo I to hydrolyze the released single-stranded DNA probe containing the sequence of Pb²⁺-DNAzyme, protecting the signal probes on the electrode surface from DNAzyme cleavage, which eventually brings about a remarkable electrochemical signal positively related to the concentration of OFL. MXene/AuNPs with excellent conductivity, large specific surface area, and good chemical stability are employed as the electrode substrate to efficiently accelerate charge transfer and increase the loading capacity for signal probes, leading to the enhancement of signal output and endowing the biosensor with high sensitivity, rapid response, and fine robustness. Taking advantage of OFL recognition–initiated Exo I hydrolysis to DNAzyme, the electrochemical platform with “signal-off/on” mode can detect OFL with good specificity and reduced background interference. Benefiting from the synergistic effect of the involved sensing elements, the developed electrochemical biosensor achieves a low detection limit of 0.47&#xa0;pg&#xa0;mL⁻¹ with a broad linear range and exhibits satisfactory detection performance in real food samples, indicating potential practical applicability in food safety monitoring.</p> Graphical Abstract <p></p>