Perfluorooctanoic acid is ubiquitous in wastewater, bioaccumulative and biotoxic, and interferes with biological wastewater treatment. Extracellular polymeric substances are important components of microbial cells. The effects of perfluorooctanoic acid (0 mg L-1, 0.1 mg L-1, 0.5 mg L-1, 1.0 mg L-1, 3.0 mg L-1, and 5.0 mg L-1) on extracellular polymeric substances were researched and the mechanisms were uncovered. The results indicated that the tightly bound extracellular polymeric substances (TB-EPS) initially increased and then decreased with the increase of perfluorooctanoic acid concentration, whereas the loosely bound extracellular polymeric substances (LB-EPS) consistently increased. Three-dimensional fluorescence spectroscopy revealed that the fluorescence intensity of TB-EPS components decreased, while that of LB-EPS components increased with the increase of perfluorooctanoic acid concentration. Fourier-transform infrared spectroscopy indicated the absorption peaks of functional groups, C=O, C-OH, C-O-C, C-N, or N-H in both TB-EPS and LB-EPS, shifted with the increase of perfluorooctanoic acid concentration. Protein secondary structure analysis demonstrated that perfluorooctanoic acid reduced the proportion of α-helices, leading to loose protein structures. Additionally, X-ray photoelectron spectroscopy showed that, as the concentration of perfluorooctanoic acid increased, the amount of C=O and O-C-O groups in LB-EPS increased and the proportion of C-(C/H) groups in TB-EPS decreased.