Herein, for the first time, a novel truncation strategy for aptamers' sequences based on molecular docking was reported for the first time. T-2 toxin and AFB aptamers were employed as models and fluorescence polarization was used to measure the affinity of aptamers. The T-2 toxin aptamer (T40) with 40 bases and the AFB aptamer with 32 bases were successfully obtained with increased affinities compared with original aptamers. Meanwhile, circular dichroism, FAM-BHQ1 dual labeled T40, enzyme digestion, and molecular dynamics simulation were performed to investigate the binding mechanism between aptamer T40 and T-2 toxin. Finally, based on MnO nanosheet and the fluorescence amplification effect of SYBR Green I, simplified and rapid fluorescence detection of T-2 toxin was achieved with a detection range of 0.03 nM to 30 nM, indicating the great potential of aptamer in practical detection applications.