A novel ratiometric electrochemical aptasensor was developed for Ochratoxin A (OTA) detection based on the binding of methylene blue (MB) to DNA with a dual signal amplification strategy. The formation of dsDNA structures between ferrocene-labeled complementary DNA (Fc-cDNA), the OTA aptamer, and complementary helper DNA (hDNA) caused Fc away from the electrode, and allowed dsDNA to bind with a certain amount of MB. Here, a small oxidation current of Fc (I_Fc) and a large oxidation current of MB (I_MB) were obtained. In the presence of OTA, its specific recognition with the aptamer induced the release of aptamer and hDNA from the electrode and subsequently the formation of hairpin structure for cDNA, which caused Fc close to the electrode and a weaker binding ability with MB. Then, an increased I_Fc and a decreased I_MB were obtained. Based on this principle, OTA could be accurately quantified by measuring the ratiometric signal of I_Fc/I_MB. Herein, the dual signal amplification strategy of the introduction of hDNA and the binding with MB after the OTA recognition was exploited to amplify the response signal. The obtained aptasensor showed a linear detection range from 10 pg mL⁻¹ to 10 ng mL⁻¹ and a detection limit of 3.3 pg mL⁻¹. The aptasensor was successfully applied to determine OTA in wheat, and the results were validated through HPLC-MS. Furthermore, by changing the target aptamers, this strategy could be universally used for the determination of various mycotoxins, showing promising potential applications for mycotoxins monitoring in agricultural products and foods.