This work proposes a voltammetric aptasensor to detect deoxynivalenol (DON) mycotoxin. The development steps of the aptasensor were partnered for the first time to a computational study to gain insights onto the molecular mechanisms involved into the interaction between a thiol-tethered DNA aptamer (80mer-SH) and DON. The exploited docking study allowed to find the binding region of the oligonucleotide sequence and to determine DON preferred orientation. A biotinylated oligonucleotide sequence (20mer-BIO) complementary to the aptamer was chosen to carry out a competitive format. Graphite screen-printed electrodes (GSPEs) were electrochemically modified with polyaniline and gold nanoparticles (AuNPs@PANI) by means of cyclic voltammetry (CV) and worked as a scaffold for the immobilization of the DNA aptamer. Solutions containing increasing concentrations of DON and a fixed amount of 20mer-BIO were dropped onto the aptasensor surface: the resulting hybrids were labeled with an alkaline phosphatase (ALP) conjugate to hydrolyze 1-naphthyl phosphate (1-NPP) substrate into 1-naphthol product, detected by differential pulse voltammetry (DPV). According to its competitive format, the aptasensor response was signal-off in the range 5.0-30.0 ng.mL(-1) DON. A detection limit of 3.2 ng.mL(-1) was achieved within a 1-hour detection time. Preliminary experiments on maize flour samples spiked with DON yielded good recovery values. (C) 2020 Elsevier B.V. All rights reserved.