The development of biosensors is gaining tremendous attention in various fields due to their extraordinary advantages, however, their sensitivity and accuracy are still challenging. Herein, we proposed a novel multifunctional nanocomposite Ce-MOF@PdNPs (MOF = metal-organic framework, PdNPs = Pd nanoparticles)-mediated triple-readout aptasensor for accurate and reliable detection of diethylstilbestrol (DES), in which Ce-MOF@PdNPs exhibited excellent peroxidase (POD)-like activity, fluormetric, and electro conductive properties. In addition, enzymes-assisted target recycling amplification was utilized to improve the sensitivity, that is the specific binding of aptamer and DES triggered an Exo III enzyme-assisted recycling reaction. The generated F-DNA was captured by the H3 strand linked to Ce-MOF@PdNPs immobilized on the electrode, exposing cleavage sites and activating the Nt.BbvCI enzyme-assisted recycling reaction, leading to the dissociation of Ce-MOF@PdNPs and a significant reduced electrochemical signal. The collected Ce-MOF@PdNPs solution also induced a proportional change in the color and fluorescence, achieving a colorimetric and fluormetric detection functionality. The detection limit under colorimetric mode was 0.16 and 0.76 ng/mL under fluorescence mode, and 0.87 pg/mL under electrochemical mode. This triple-readout aptasensor exhibits high sensitivity, selectivity and accuracy, providing a new idea for designing novel biosensing platforms for veterinary drug residue detection.