The highest removal efficiencies of COD and TN were achieved under 10 mg/L of Al3+. The highest TP removal efficiency occurred under 30 mg/L of Al3+. EPS, PS and PN concentrations increased with the addition of Al3+. Sludge properties significantly changed with the addition of Al3+. Aluminum ions produced by aluminum mining, electrolytic industry and aluminum-based coagulants can enter wastewater treatment plants and interact with activated sludge. They can subsequently contribute to the removal of suspended solids and affect activated sludge flocculation, as well as nitrogen and phosphorus removal. In this study, the effects of Al3+ on pollutant removal, sludge flocculation and the composition and structure of extracellular polymeric substances (EPS) were investigated under anaerobic, anoxic and oxic conditions. Results demonstrated that the highest chemical oxygen demand (COD) and total nitrogen (TN) removal efficiencies were detected for an Al3+ concentration of 10 mg/L. In addition, the maximal dehydrogenase activity and sludge flocculation were also observed at this level of Al3+. The highest removal efficiency of total phosphorus (TP) was achieved at an Al3+ concentration of 30 mg/L. The flocculability of sludge in the anoxic zone was consistently higher than that in the anaerobic and oxic zones. The addition of Al3+ promoted the secretion of EPS. Tryptophan-like fluorescence peaks were detected in each EPS layer in the absence of Al3+. At the Al3+ concentration of 10 mg/L, fulvic acid and tryptophan fluorescence peaks began to appear, while the majority of protein species and the highest microbial activity were also detected. Low Al3+ concentrations (<10 mg/L) could promote the removal efficiencies of COD and TN, yet excessive Al3+ levels (>10 mg/L) weakened microbial activity. Higher Al3+ concentrations (>30 mg/L) also inhibited the release of phosphorus in the anaerobic zone by reacting with PO43-.