Due to the difficulties in monoclonal antibody production specific to mycotoxins, aptameric probes have been considered as suitable alternatives. The low efficiency of the SELEX procedure in screening high affinity aptamers for binding mycotoxins as small molecules can be significantly improved through computational techniques. Previously, we designed five new aptamers to aflatoxin B1 (AFB1) based on a known aptamer sequence (Patent: PCT/CA2010/001 292, Apt1) through a genetic algorithm-based in silico maturation strategy and experimentally measured their affinity to the target toxin. Here, integrated molecular dynamic simulation (MDs) studies with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis to clarify the binding modes, critical interacting nucleic bases and energy component contributions in the six AFB1-binding aptamers. The aptamer F20, which was selected in the first work, showed the best free binding energy and complex stability compared to other aptamers. The trajectory analysis revealed that AFB1 recognized F20 through the groove binding mode along with precise shape complementarity. The MD simulation results revealed that dynamic water intermediate interactions also play a key role in promoting complex stability. According to the MM-PBSA calculations, van der Waals contacts were identified as dominant energy components in all complexes. Interestingly, a high consistency is observed between the experimentally obtained binding affinities of the six aptamers with their free energy solvation. The computational findings, confirmed via previous experiments, highlighted the binding modes, the dynamic hydration of complex components and the total free interacting energy as the crucial criteria in discovering high functional aptameric probes.