In this study, the flocculation and sedimentation performance of thermophilic activated sludge with single and multiple dosing of trivalent aluminum (Al3+) were studied. The thermophilic activated sludge samples were cultivated in sequencing batch reactors at 55 degrees C. The dosages of Al3+ were 0.125, 0.5, 1.0, 1.5, and 2.0 meq/L for single dosing, and 0.1 meq/L for multiple dosing. The experimental results indicated that single Al3+ dosing could enhance sludge flocculation, but deteriorate sedimentation. While multiple dosing, which allows sufficient time for sludge restructuring and floc aggregation, could optimize flocculation and maintain sedimentation simultaneously. LB-EPS played a more important role than interaction energy in the improvement. Although the responding of zeta potential was insignificant, better flocculability and settleability were achieved through Al3+ dosing.