To enhance the sensitivity of an aptasensor, a novel strategy was designed to develop an electrochemical aptasensor based on poly(3,4-ethylenedioxy thiophene)-gold nanoflower (PEDOT-AuNF) composites supported on a three-dimensional graphene oxide sponge (GOS). GOS with a three-dimensional sponge-like porous structure, exhibiting excellent electrical conductivity and a large surface area, provided the first amplification of the electrochemical signal for ochratoxin A (OTA) detection. PEDOT-AuNFs, synthesized by an ionic liquid-assisted one-pot method, presented a peculiar hierarchical flower-like structure, a high electroactive surface area, and more binding sites for immobilizing the aptamer molecules by the Au-S bonds. When PEDOT-AuNFs were supported on the surface of GOS by the interaction of the π-π packing between PEDOT and graphene oxide, a synergistic effect was produced to provide the second amplification for the aptasensor. PEDOT-AuNFs/GOS provided an ultrasensitive detection technique by multiple signal amplification for the electrochemical sensing of OTA. Consequently, this strategy not only endowed the aptasensor with high sensitivity but also needed no complicated signal amplification. The electrochemical sensor was fabricated successfully on a glassy carbon electrode to detect OTA with a linear response in the range of 0.01-20 ng L and a limit of detection of 4.9 pg L. Moreover, it displayed good specificity, reproducibility and stability. The utilization of the proposed aptasensor for the quantitative determination of OTA in wine indicates that it can find promising applications in detecting OTA and even other mycotoxins in foodstuffs.