Abstract: Ochratoxin A is a kind of common fungal toxin that mainly contaminates food crops and their products. It is stable and not easily degraded when residing in food. Toxicological studies have shown that OTA is harmful to human and animal health since it has strong hepatotoxicity, nephrotoxicity, neurotoxicity, and teratogenicity. To ensure food safety and human health, it is required to establish simple and rapid detection methods. In this experiment, a fluorescent sensor based on DNA aptamer was constructed for the rapid detection of OTA in food. A simple and rapid fluorescent sensor for OTA detection was constructed by using the aptamer with carboxyl fluorescein (FAM) and the complementary chain with black hole quenching group (BHQ1). When OTA is absent, Apt-FAM binds to cDNA-BHQ1 by base complementary pairing, the fluorescent group FAM is close to the quenching group BHQ1, and the signal of fluorescent group FAM is suppressed by the effect of fluorescence resonance energy transfer. When OTA is present, OTA binds to Apt-FAM to form G-quadruplex, which keeps away from cDNA-BHQ1, and the fluorescent signal is restored. By controlling the distance between the FAM fluorophore and the quenching group BHQ1, the intensity of the fluorescence signal is changed, thereby realizing the detection of OTA. In order to optimize the detection performance of the sensor, the number of bases and complementary positions of complementary strands, the total reaction time, the ratio of cDNA to Apt dosage, and the pH of the reaction system were optimized. Through the optimization of experimental conditions, the optimal complementary strand was cDNA1-BHQ1, the optimal reaction time was 60 min, the optimal dosage ratio of cDNA to Apt was 1∶1, and the pH value of the optimal reaction system was 7.4. In the range of OTA concentration from 0.5 ng/mL to 100 ng/mL, the relative fluorescence intensity and the OTA concentration were logarithmic, showing a good linear relationship. The linear equation is y=31.934 98+41.714 94 lg(C_OTA), and the correlation coefficient R²=0.997, with a detection limit of 0.13 ng/mL. The experimental results showed that in the evaluation results of anti-interference performance of OTA and other toxins, the fluorescence sensor had good specificity for detecting OTA. The sensor also had good reproducibility. The prepared fluorescent sensor was used for spiked detection of corn samples and wine samples, and the average recoveries were 100.0%-102.0% and 98.0%-102.2% respectively, indicating that the sensor can be used for the detection of OTA in actual samples. In conclusion, a simple and fast fluorescent aptamer sensor for high sensitivity and effective detection of OTA has been successfully designed, which is of great significance for ensuring food safety.