T-2 toxin is a highly poisonous trichothecene and a well-known contaminant in cereal grains and cereal-based products, hence it is essential to design a simple, reliable, and precise detection mechanism for ensuring food safety and human health. In response to this imperative, we innovatively designed an unreported split T-2 toxin aptamer probe, and the cooperative binding mechanism of the split aptamer to T-2 was first investigated. Based on these, a dual-mode fluorescence (FL) and fluorescence polarization (FP) aptasensor was developed for T-2 toxin detection in peanut and beer samples. The results showed an excellent detection property of the aptasensor, indicating a linear range of 0.1-20 nM and 0.2-50 nM at a detection limit of 0.1 nM, 0.12 nM for FL and FP signals, respectively. In actual sample assays, recoveries of 101.7% -115.2%, 109.8% -121.6% for FL and FP mode, respectively, were reached in peanuts, whereas recoveries ranged from 81.0% -124.0%, 94.1% -107.8% for FL and FP mode in beer. This designed dual-mode biosensor not only showed excellent performances but also verified each other and proved the reliability of binary split aptamer molecular probes.