Abstract: Aflatoxin B₁ (AFB₁) is a typical fungal toxin residual in agricultural products and poses a great threat to both humans and animals when it accumulates to a certain amount, therefore, accurate and rapid determination of AFB₁ is of great importance for food safety. In this study, an X structure based on DNA nanotechnology was designed and a fluorescent aptamer sensor for the highly sensitive detection of AFB₁ was constructed by combining magnetic separation technology. In this system, two DNA strands were firstly used to form an X-skeleton and immobilized on magnetic beads, and a fluorescent group-labeled aptamer strand was used as the signal probe (FAM-Apt). When AFB₁ is not present, FAM-Apt can form a double-stranded structure by base complementary pairing with the four ends of the X-skeleton, followed by magnetic separation and aspiration of the supernatant for fluorescence detection to obtain a low background signal. When AFB₁ is present, FAM-Apt preferentially binds to AFB₁ due to the high specificity and affinity of the target and the aptamer, and is detached from the sensor and free in the supernatant, which is detected after magnetic separation to obtain a higher fluorescence signal. Finally, the rapid detection of AFB₁ was achieved according to the change of fluorescence signal intensity in the supernatant. Under the optimal experimental conditions, the relative fluorescence intensity of the sensor was linearly correlated with lg(ρ _AFB1) in the range of 0.05-100 ng/mL (R²=0.99), and the detection limit was as low as 9 pg/mL. Satisfactory results were obtained by applying the designed sensor platform to spiked samples for recovery testing.