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A magnetic beads (MBs)–assisted fluorescence aptasensing approach based on dual DNA tweezers and magnetic separation was established for the detection of ochratoxin A (OTA) and fumonisin B1 (FB1). A dual DNA tweezers structure with four ends linked with fluorophores (FAM, ROX) and quenchers (BHQ1, BHQ2) was designed, and produced the high initial fluorescence signals because of the long spatial distance between FAM and BHQ1, ROX, and BHQ2. Bio-aptamer/anti-aptamer of OTA and bio-aptamer/anti-aptamer of FB1 were respectively annealed to form dsDNA, and immobilized to MBs coated with streptavidin (SA). With the existence of OTA and FB1, OTA and FB1 preferentially bound with their respective bio-aptamers, which made anti-aptamers dissociate from dsDNA coupled on MBs. After magnetic separation, the dissociated anti-aptamers reacted with dual DNA tweezers, respectively, which made DNA tweezers close and the fluorescence was quenched. The linear ranges of approach for OTA and FB1 detection were 0.05–20 ng/mL and 0.1–40 ng/mL, respectively. The limit of detection for OTA and FB1 was 0.029 ng/mL and 0.061 ng/mL. The prepared MBs-assisted fluorescence aptasensing approach was applied to detect OTA and FB1 in spiked red wine and corn samples, which showed good recoveries between 92 and 106%. [ABSTRACT FROM AUTHOR]