Aflatoxins (AFs) are the carcinogenic secondary metabolites produced by fungi such as Aspergillus flavus, A. parasiticus, and A. nomius. These fungi are widespread and have severely contaminated food/feed supplies. The major health consequences of consuming AF-contaminated food/feed are Hepatocellular carcinoma and its associated complications. Therefore, detecting and quantifying AFs in foods and feeds is critical to achieve food safety. Among the available methods, aptamer-based techniques are receiving more attention considering their specificity, sensitivity, low cost, and ease of production and handling. Though AFB₁-specific aptamers have been well studied, ligand–aptamer molecular interactions are not clear. In the present study, the three-dimensional structures of available AFB₁-specific DNA aptamers were predicted using RNA-based web servers with an automated machine translation principle and some manual changes in the script file. Molecular docking was performed between selected DNA aptamers and AFB₁. Molecular docking results revealed that the AFB₁ was located inside the pocket area of the predicted tertiary structures and formed electrostatic interactions with the nucleotides of aptamers. The binding affinities of all aptamers were between -7.3 and -11.7 kcal/mol. This study adds to our understanding of how AFB₁ interacts with all available aptamers. The developed workflow enables the generation and examination of additional appealing ligand–aptamer complexes to detect AF and other mycotoxins.