(OTA) is positively correlated with an increased risk of developing cancer in nephrotoxic and hepatotoxic patients. Therefore, it is of great significance for the highly sensitive, highly selective, and timely detection of OTA. We described here an electrochemical aptasensor for OTA analysis, which took advantage of the favorable properties of gold nanoparticles (AuNPs) functionalized zinc oxide (ZnO) composites and the intercalative binding between methylene blue (MB) and nucleic acid. There were two label-free aptamers: one to capture OTA and another serving as complementary DNA (cDNA), enabling connection to the ZnO-Au composite’s immobilized electrode. Once OTA was present, the aptamer could capture OTA and detach from the electrode interface, thus, preventing MB from accessing electrode surface for efficient electron transfer; a decreased peak current was monitored by differential pulse voltammetry. The aptasensor presented nice analytical performance for OTA detection in the range of 0.1−30,000 pg·mL−1, with a detection limit of 0.05 pg·mL−1. Moreover, the developed biosensor could be applied to actual sample (wine and beer) analysis.