Colorimetric aptasensor based on assembly of salt-induced gold nanoparticles (AuNPs) is a promising biosensor. However, the molecular mechanism of the aptasensor is far from being fully understood. Herein, molecular dynamics (MD) simulation was used to investigate molecular interactions in the detection of ochratoxin A (OTA) including the following: (i) the molecular recognition of the anti-OTA aptamer, (ii) OTA-aptamer interactions in monovalent (Na+) and divalent (Mg2+) electrolytes, (iii) the binding mode of citrate on the AuNP surface, (iv) interactions of the aptamer with citrate-capped AuNPs, and (v) a detailed mechanism of the aptasensor. Our MD simulations revealed a specific binding of the OTA-aptamer complex, compared with OTB and warfarin. Compared with Na+, Mg2+ ions exerted a more effective attractive force between OTA and anti-OTA aptamer. Three different binding modes of citrate on AuNP surfaces were found. The kinetics of the adsorption of unfolded aptamers onto the citrate-capped AuNP was also elucidated. Most importantly, our MD simulation revealed an insightful analysis of the molecular mechanisms in the AuNP-based aptasensor and paved the way for the design of a novel colorimetric aptasensor for other target molecules, which is not limited to OTA detection.