Ultrasonic pretreatment of excess sludge can improve its aerobic digestibility, leading to enhanced sludge reduction. In order to understand the mechanisms of this improvement, sludge flocs were divided into four layers, i.e. (1) slime, (2) loosely bound extracellular polymeric substances (LB-EPS), (3) tightly bound EPS (TB-EPS) and (4) pellet. Extracellular proteins, polysaccharides and five types of hydrolytic enzymes (protease, alpha-amylase, alpha-glucosidase, alkaline-phosphatase and acid-phosphatase) from sludge flocs were investigated to determine their influence on sludge aerobic digestion after ultrasonic pretreatment. Results suggested that most of the extracellular enzymes (except alpha-amylase) were present in pellet and TB-EPS layers, with minor quantities detected in LB-EPS and slime layers, and almost none detected in bulk solution. As for alpha-amylase in sludge flocs, most of it (52.6%) was also mainly bound with pellet; however, the rest of it was dispersed nearly uniformly throughout the sludge flocs. Ultrasonic pretreatment enhances enzymatic activities and promotes the shifts of extracellular proteins, polysaccharides and enzymes from inner layers of sludge flocs, i.e., pellet and TB-EPS, to outer layers, i.e., slime, to increase the contact and interaction among extracellular proteins, polysaccharides and enzymes that were originally embedded in the sludge flocs, resulting in improved efficiency in aerobic digestion. The optimum ultrasonic pretreatment conditions had a lasting time of 10min and density of 3 kWL(-1) at the frequency of 20 kHz. With the optimum ultrasonic pretreatment, the sludge reduction for TSS in aerobic digestion was 42.7% in which the part of 11.8% was removed by the ultrasonic pretreatment, compared with 20.9% for control, after an aerobic digestion time of 10.5d.