Abstract: Rapid and accurate detection of Aspergillus flavus is crucial for preventing fungal contamination of grain, controlling aflatoxin production in grains, and ensuring safety of grain storage. In this study, specific primers targeting the OMT-1 gene of A. flavus were designed and a SYBR Green I qPCR-based detection system was established. We determined a relationship between the copy number of the OMT-1 gene in A. flavus producing aflatoxin and the CT value, and verify the absolute quantitative method of qPCR for Aspergillus flavus. This method demonstrated high specificity and sensitivity, with a detection limit of 0.015 μg/mL. The logarithm of copy number of OMT-1 gene is linearly correlated with CT value, with a standard curve equation of y=-3.519 2x+55.905 (R²=0.997 3) and an amplification efficiency of 107.0%. Additionally, the copy number of the OMT-1 gene showed a strong correlation with the colony count data obtained from the same set of samples (R²=0.97). These results indicate that the qPCR absolute quantification method could be used to monitor the expression of the A. flavus OMT-1 gene in maize.