Aluminum flocculant can enhance the flocculating performance of activated sludge. However, the binding mechanism of aluminum ion (Al(3+)) and extracellular polymeric substances (EPS) in activated sludge is unclear due to the complexity of EPS. In this work, three-dimensional excitation emission matrix fluorescence spectroscopy (3DEEM), fluorescence quenching titration and Fourier transform infrared spectroscopy (FT-IR) were used to explore the binding behavior and mechanism between Al(3+) and EPS. The results showed that two fluorescence peaks of tyrosine- and tryptophan-like substances were identified in the loosely bound-extracellular polymeric substances (LB-EPS), and three peaks of tyrosine-, tryptophan- and humic-like substances were identified in the tightly bound-extracellular polymeric substances (TB-EPS). It was found that these fluorescence peaks could be quenched with Al(3+) at the dosage of 3.0 mg/L, which demonstrated that strong interactions took place between the EPS and Al(3+). The conditional stability constants for Al(3+) and EPS were determined by the Stern-Volmer equation. As to the binding mechanism, the -OH, N-H, C=O, C-N groups and the sulfur- and phosphorus-containing groups showed complexation action, although the groups in the LB-EPS and TB-EPS showed different behavior. The TB-EPS have stronger binding ability to Al(3+) than the LB-EPS, and TB-EPS play an important role in the interaction with Al(3+).