Abstract: Ochratoxin A is a mycotoxin produced by Penicillium and Aspergillus. Ochratoxin A is widely polluted in food and since it is thermal stable and not easily to be degraded, it is harmful for the health of humans and livestock, and also may cause serious food safety problems. Therefore, it is urgent to establish a sensitive and selective method for the rapid detection of OTA in food. In order to achieve convenient and rapid detection of ochratoxin A (OTA), a simple, low-cost and easy long-term storage paper-based biosensor was constructed by using the fluorescence resonance energy transfer (FRET) between carboxyfluorescein (FAM) and black hole quencher group (BHQ1) and using a paper-based substrate as the sensor substrate. An aptamer that specifically recognized the OTA (Apt-FAM) was attached to the paper base by a strong interaction between biotin and streptavidin. When OTA was present, OTA preferentially bound to Apt-FAM, and the complementary strand (cDNA-BHQ1), which was complementary to Apt-FAM, could not be attached to the paper base by base complementary pairing, and the quencher group BHQ1 was far away from the fluorescent group FAM, thus obtaining a stronger value of FAM fluorescence signal. When OTA was not present, cDNA-BHQ1 was bound to Apt-FAM through base complementary pairing and attached to the surface of the paper-based sensor, and the quencher group BHQ1 was close to the fluorophore FAM and suppressed the signal of the fluorophore FAM by fluorescence resonance energy transfer, which decreased the fluorophore FAM signal response value. Under the optimal detection conditions, the linear range of the sensor to detect OTA was 10 ng/mL to 2 000 ng/mL, the linear equation was y=-15.858 46+65.631 38lg(C_OTA) (R²=0.995), and the minimum detection limit was 1.2 ng/mL. The paper-based biosensor prepared in this experiment had excellent reproducibility and stability, and could be stored for a long time at 4℃ under light-proof condition, which had certain practical application value. The paper-based biosensor has been successfully used to detect OTA in rice flour and mung bean pastry samples with the average recoveries of 89.2%-111.0% and 89.0%-103.0%, respectively. In conclusion, this study successfully constructed a paper-based biofluorescent sensor for rapid detection of ochratoxin A in food, which provides a reference for achieving rapid detection of mycotoxins in the field of food safety.