A simple and ultrasensitive method was developed for the detection of Ochratoxin A, utilizing an aptamer as a molecular recognition probe and real-time quantitative PCR (RT-qPCR) amplification of its complementary DNA as signal generators. Under the optimized conditions, the cycle threshold (Ct) increased linearly with 10-fold serial dilutions of Ochratoxin A (OTA) from 5×10⁻⁶ to 5 ng mL⁻¹, with a limit of detection (LOD) of 1 fg mL⁻¹. The specificity of this aptasensor was considered to be excellent, as when tested against four other toxins it produced no obvious Ct value change. Furthermore, a satisfactory analyte concentration recovery was obtained from a series of concentrations of OTA spiked into red wine. Therefore, this highly sensitive approach shows a significant potential in a wide range of target analytes.