Some surfactants can enhance the removal of phenol by laccase (Lac) in various industrial effluents. Their behavior and function in the biodegradation of phenolic wastewater have been experimentally reported by many researchers, but the underlying molecular mechanism is still unclear. Therefore, the interaction mechanisms of phenol with Lac from Trametes versicolor were investigated in the presence or absence of Triton X-100 (TX100) or rhamnolipid (RL) by molecular docking and molecular dynamics (MD) simulations. The results indicate that phenol contacts with an active site of Lac by hydrogen bonds (HBs) and van der Waals (vdW) interactions in aqueous solution for maintaining its stability. The presence of TX100 or RL results in the significant changes of enzymatic conformations. Meanwhile, the hydrophobic parts of surfactants contact with the outside surface of Lac. These changes lead to the decrease of binding energy between phenol and Lac. The migration behavior of water molecules within hydration shell is also inevitably affected. Therefore, the amphipathic TX100 or RL may influence the phenol degradation ability of Lac by modulating their interactions and water environment. This study offers molecular level of understanding on the function of surfactants in biosystem.