© 2015 American Chemical Society. In this article, a novel aptasensor for ochratoxin A (OTA) detection based on a screen-printed carbon electrode (SPCE) modified with polythionine (PTH) and iridium oxide nanoparticles (IrO<inf>2</inf> NPs) is presented. The electrotransducer surface is modified with an electropolymerized film of PTH followed by the assembly of IrO<inf>2</inf> NPs on which the aminated aptamer selective to OTA is exchanged with the citrate ions surrounding IrO<inf>2</inf> NPs via electrostatic interactions with the same surface. Electrochemical impedance spectroscopy (EIS) in the presence of the [Fe(CN)<inf>6</inf>]<sup>-3/-4</sup> redox probe is employed to characterize each step in the aptasensor assay and also for label-free detection of OTA in a range between 0.01 and 100 nM, obtaining one of the lowest limits of detection reported so far for label-free impedimetric detection of OTA (14 pM; 5.65 ng/kg). The reported system also exhibits a high reproducibility, a good performance with a white wine sample, and an excellent specificity against another toxin present in such sample.