In this article, the interaction mechanism between the superoxide dismutase (SOD) and the triclosan (TCS), a kind of antimicrobial agent which is of widely application with potential effects both on environment and human health, was explored through a series of spectroscopic methods, animal experiment and the molecular docking simulation. The negative free energy change ∆G, enthalpy change (∆H = 162.21 kJmol⁻¹) and entropy change (∆S = 615 Jmol⁻¹K⁻¹) demonstrated that TCS could combine with SOD spontaneously through hydrophobic interaction to form a complex. The binding constants of K_a293 and K_a313 were 1.706 × 10³ and 1.2 × 10⁵ Lmol⁻¹, respectively. Furthermore, the interaction could also influence the skeleton structure and secondary contents of SOD. The molecular docking analysis revealed the TCS located between two subunits of SOD, and there was a hydrogen bond between TCS and the residue Asn51 of SOD, which influenced the structure of protein and resulted in a decrease of enzyme activity. This work could help understand the interaction mechanism between SOD and TCS. Moreover, it could also be used to consult for toxicity assessment of TCS at molecular level.