Abstract: A biosensor for aflatoxin B₁ (AFB₁) was constructed by using nucleic acid aptamer as the specific recognition element and SYBR Green I (SGI) fluorescent dye as the signal output unit. The test conditions were optimized, revealing the following optimal parameters: the molar ratio of the complementary chain of the aptamer to the aptamer was 1.5, the addition amount of SGI was 10 μL, the interaction time of the double-chain aptamer with SGI was 2 min, and the interaction time of the aptamer with AFB₁ was 14 min. As shown by the results, when the mass concentration of AFB₁ ranged from 0.1 μg · L⁻¹ to 1 000 μg · L⁻¹, the change in fluorescence intensity exhibited a linear relationship with the logarithm of its mass concentration, with detection limit (3S/N) of 0.081 μg · L⁻¹. Test for recovery was made on actual corn samples by the standard addition method, giving results in the range of 95.2%—105%, with RSDs of the determined values (n=7) less than 6.0%. Compared to other aptamer sensors, this fluorescence aptamer sensor exhibited numerous advantages in AFB₁ detection, including simplicity of operation, a wide detection range, high sensitivity, strong specificity, and low cost. Consequently, it was well-suited for rapid on-site determination.