An aptamer to selectively detect ochratoxin A (OTA) in cereals was developed using silver nanoparticles (AgNPs) in combination with reduced graphene oxide (rGO). As a result of characterization experiments conducted in this study, AgNPs were confirmed to be polydisperse. The electrocatalytic activity was achieved by immobilizing OTA-bovine serum albumin (OTA-BSA) on the rGO/AgNPs substrate. On the basis of the experimental conditions optimized for the aptasensor, the linear dynamic range was 0.002−0.016 mg/L and the threshold was 7 × 10 −4 mg/L (S/N = 3). At an atomic and molecular level, computational adsorption studies revealed how OTA-BSA interacts with the rGO/AgNPs composite substrates on a spatial scale. Calculations using density functional theory (DFT) revealed that OTA has an energy gap of −4.5 eV, which implies a strong tendency to operate as an electron donor. The use of computational modeling has also provided structural information about interactions between biomolecules and nanostructures, as well as the electronic properties of OTA. The electrochemically modified aptasensor showed good agreement with computed adsorption energies and current responses. In addition to its excellent reproducibility and good stability, the proposed aptasensor demonstrated its applicability in the food industry.