<p>Ochratoxin A (OTA), a highly toxic mycotoxin, extensively contaminates a wide range of food commodities. Given increasing demands for sensitive and user-friendly detection platforms, we developed a colorimetric OTA biosensor leveraging copper-silver nanoclusters (Cu/Ag NCs), whose peroxidase-like activity is augmented via G/C-rich DNA hybridization. The system employs two tailored DNA strands: a template strand featuring a cytosine-rich domain for NC synthesis flanked by terminal recognition segments and an enhancer strand containing a guanine-rich signal amplification domain and a complementary linker. Upon OTA binding with the aptamer, the apt-template complex dissociates, facilitating template-enhancer hybridization to form G/C-rich double-stranded DNA. This structural transition substantially enhances the peroxidase-like activity of DNA-Cu/Ag NCs, accelerating the oxidation of 3,3′,5,5′-tetramethylbenzidine by H₂O₂ while simultaneously amplifying the colorimetric signal. Quantitative OTA detection is accomplished by monitoring this dynamic color response. The biosensor demonstrates a semi-log linear detection range from 5.0&#xa0;nM to 1.0&#xa0;μM with a detection limit of 5.0&#xa0;nM. Furthermore, the specific test has high selectivity for other common mycotoxins, and its applicability has been validated through real sample analysis, suggesting that the DNA-Cu/Ag NCs biosensor used for OTA detection holds promising practical applications in food samples.</p> Graphical abstract