The control of interfacial microbial pollution is of great significance for water safety. Herein, the tribo-catalysis ability of zinc oxide (ZnO) has been investigated, which can realize the control of tightly-bound extracellular polymeric substances (T-EPS) in water under dark environment. The DFT calculation proves the Fe doping introduces the impurity level and decreases the work function from 5.071 eV to 5.045 eV, improves the charge separation of ZnO, and eventually enhances the catalytic reaction efficiency. Characterizing the catalytic reaction process by three-dimensional fluorescence (3D EEM) and fluorescence regional integration (FRI) method, it is found that the T-EPS solution can be degraded 75.8% by Fe-ZnO in 12 min, while ZnO can only degrade 32.2%. Combining with high-resolution scanning probe microscope (HR-SPM) and attenuated total reflection method (ATR-FTIR), hydration layers consist with hydroxyl layer (∼0.23 nm) and water molecular layer (∼0.27 nm) are observed at the interface between Fe-ZnO and T-EPS solution, and terminal hydroxyl group (OHt) is considered to be the active site for the generation of radicals. This study provides an idea for exploring the mechanism of tribo-catalytic reaction and shows its application prospect in the field of microbial inhibition in water.