Understanding the interactions of drug molecules with biomacromolecules at a micro-scale level is essential to design potent drugs for the treatments of human genome diseases. To unravel the mechanism of binding of aristololactam-β-D-glucoside (ADG) and phenylalanine transfer RNA (tRNAPhe), an integrated computational strategy combining quantum mechanics (QM) calculation, molecular docking and atomistic molecular dynamics (MD) simulation was present in this work. QM calculations were performed to derive the partial charges of ADG, molecular docking was used to determine the binding poses of ADG on the tRNAPhe, and atomistic MD simulations were conducted to examine the thermal stability of five predicted binding poses for the complex of ADG and the tRNAPhe. The binding free energies of the five complexes were then calculated using the molecular mechanics/generalized born surface area approach with the variable internal dielectric constant model. By comparing computed affinities and experimentally-measured values in the binding free energy, we identified a most likely binding structure of ADG and the tRNAPhe. Further analysis of energy of the ADG-tRNA complex revealed that the aristololactam of ADG provides binding specificity to the tRNAPhe, and the D-glucoce contributes to the affinity for binding with the tRNAPhe.