An aptamer sensor based on manganese dioxide (MnO₂) nanosheets was developed for the detection of zearalenone (ZEN). The ZEN aptamer was modified at the 5′-end by a 6-carboxyfluorescein (6-FAM) fluorophore with self-assembly on MnO₂ nanosheets. Interaction of the 6-FAM fluorophore at the 5′-end of the ZEN aptamer with the MnO₂ nanosheet lowered fluorescence (FL) intensity due to fluorescence resonance energy transfer (FRET). The introduction of ZEN into the sensing system resulted in hybridization with the ZEN aptamer, forming a stable G-quadruplex/ZEN, which exhibited a low affinity for the MnO₂ nanosheet surface. The distance between the 6-FAM fluorophore and MnO₂ nanosheet hampered FRET, with a consequent strong FL signal. Under the optimal experimental conditions, the FL intensity of the sensing system showed a good linear correlation with ZEN concentration in the range of 1.5–10.0 ng mL⁻¹, and a detection limit (S/N = 3) of 0.68 ng mL⁻¹. The sensing system delivered enhanced specificity for the detection of ZEN, and can find wide application in the detection of other toxins by replacing the sequence of the recognition aptamer.