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Mycotoxins posit serious threats to human and animal health, and numerous efforts have been performed to detect the multiple toxins by a single diagnostic approach. To best of our knowledge, for the first time, we synthesized an aptamer induced “turn on” fluorescence resonance energy transfer (FRET) biosensor using dual-color gold nanoclusters (AuNCs), l-proline, and BSA synthesized AuNCs (Lp-AuNCs and BSA-AuNCs), with WS2 nanosheet for simultaneous recognition of aflatoxinB1 (AFB1) and zearalenone (ZEN) by single excitation. Here, AFB1 aptamer stabilized blue-emitting AuNCs (AFB1-apt-Lp-AuNCs) (at 442 nm) and ZEN aptamer functionalized with red-colored AuNCs (ZEN-apt-BSA-AuNCs) (at 650 nm) were employed as an energy donor and WS2 nanosheet as a fluorescence quencher. With the addition of AFB1 and ZEN, the change in fluorescence intensity (F.I) was recorded at 442 and 650 nm and can be used for simultaneous recognition with a detection limit of 0.34 pg mL–1 (R 2 = 0.9931) and 0.53 pg mL–1 (R 2 = 0.9934), respectively. Most importantly, the semiquantitative determination of AFB1 and ZEN can also be realized through photovisualization. The current approach paves a new way to develop sensitive, selective, and convenient metal nanocluster-based fluorescent “switch-on” probes with potential applications in multipurpose biosensing.

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