TLR7 and TLR8 are key members of the Toll-like receptor family, playing crucial roles in the signaling pathways of innate immunity, and thus become attractive therapeutic targets of many diseases including infections and cancer. Although TLR7 and TLR8 show a highly degree of sequence homology, their biological response to small molecule binding is very different. Aiming to understand the mechanism of selective profiles of small molecule modulators against TLR7 and TLR8, we carried out molecular dynamic simulations on three imidazoquinoline derivatives bound to the receptors separately. They are Resiquimod (R), Hybrid-2 (H), and Gardiquimod (G), selective agonists of TLR7 and TLR8. Our MD trajectories indicated that in the complex of TLR7-R and TLR7-G, the two chains forming the TLR7 dimer tended to remain “open” conformation, while the rest systems maintained in the closed format. The agonists R, H, and G developed conformational deviation mainly on the aliphatic tail. Furthermore, we attempted to quantify the selectivity between TLR7 and TLR8 by binding free energies via MM-GBSA method. It showed that the three selected modulators were more favorable for TLR7 than TLR8, and the ranking from the strongest to the weakest was H, R and G, aligning well with experiment data. In the TLR7, the flexible and hydrophobic aliphatic side chain of H has stronger van der Waals interactions with Val381 and Phe351 but only pick up interaction with one amino acid residue i.e. Tyr353 of TLR8. Unsurprisingly, the positively charged side chain of G has less favor interaction with Ile585 of TLR7 and Val573 of TLR8 explaining G is weak agonist in both TLR7 and TLR8. All three imidazoquinolines can form stable hydrogen bonds with Asp555 of TLR7 and the corresponding Asp543 of TLR8. In brief, the set of total 400ns MD studies sheds light on the potential selective mechanisms of agonists towards TLR7 and TLR8, indicating the van der Waals interaction as the driving force for the agonists binding, thus provides us insights for more potent and selective modulators to cooperate with the hydrophobic nature of the binding pocket.