On the basis of Tb(3+), structure-switching aptamer and magnetic beads (MBs), a signal-on fluorescent aptasensor was developed for the label-free determination of OTA in wheat. Initially, the specific sequence of the anti-OTA aptamer labeled with a biotin group, was attached to streptavidin-modified MBs. Two single-stranded signal probes were added and naturally hybridized with anti-OTA aptamer to form the duplex structure in the solution. Due to the fact that single-stranded oligonucleotides can greatly enhance the emission of Tb(3+) in solution but duplexes do not, through magnetic separation, the supernatant liquid of the above solution contained no single-stranded DNA and cannot increase the emission of Tb(3+). While upon OTA addition, it will bind with aptamer to form OTA-aptamer G-quadruplex while releasing two single-stranded signal probes. Through magnetic separation, the released single-stranded signal probes left in the supernatant liquid can dramatically increase the fluorescent intensity of Tb(3+). By employing the above strategy, this aptasensor can detect as low as 20 pg/mL OTA with high specificity. To the best of our knowledge, the proposed aptasensor is the first attempt to use the fluorescent characteristics of Tb(3+) for OTA detection, which may represent a promising path toward routine quality control of food safety.