<p>Chloramphenicol (CAP), a broad-spectrum antibiotic derived from <i>Streptomyces</i>, readily persists and accumulates in humans through pathways such as the food chain, leading to significant toxic effects. Herein, a novel electrochemiluminescence (ECL) aptasensor based on the signal “ON–OFF–ON” strategy is proposed for CAP detection in food. We utilized the immobilization capacity and enzyme-mimicking activity of iron-lanthanum doped prussian blue analogue (Fe-La PBA) to catalytically enhance the luminol–H₂O₂ luminescent system, thereby engineering the composite nanozyme-based luminophore (luminol-Au@Fe-La PBA). Moreover, polydopamine (PDA)-coordinated cadmium sulfide (CdS) decorated with gold nanoparticles (Au@CdS/PDA) serves as an energy acceptor to quench the signal from emitter luminol-Au@Fe-La PBA. Firstly, the luminol-Au@Fe-La PBA emitter was immobilized on a glassy carbon electrode (GCE), providing a strong initial ECL signal and serving as a platform for anchoring cDNA hybridized with the aptamer (Apt). Then the specific quenching probe (Apt-Au@CdS/PDA) was introduced, leading to substantial signal reduction. Upon the action of CAP, the dissociation of the Apt–cDNA hybrid duplex led to the detachment of the quenching probe from the electrode surface, thereby restoring the ECL signal intensity. The aptasensor demonstrated a linear response over a concentration range of 10 to 10⁷ pg mL^− 1, and LOD was 1.18 pg mL^− 1, exhibiting satisfactory recoveries in various food samples and quality control samples.</p> Graphical Abstract <p></p>