Effect of ultrasonic pretreatment on sludge dewaterability was determined and the fate of extracellular polymeric substances (EPS) matrix in mesophilic anaerobic digestion after ultrasonic pretreatment was studied. Characteristics of proteins (PN), polysaccharides (PS), excitation-emission matrix (EEM) fluorescence spectroscopy and molecular weight (MW) distribution of dissolved organic matters (DOM) in different EPS fractions were evaluated. The results showed that after ultrasonic pretreatment, the normalized capillary suction time (CST) decreased from 44.4 to 11.1 (sec x L)/g total suspended solids (TSS) during anaerobic digestion, indicating that sludge dewaterability was greatly improved. The normalized CST was significantly correlated with PN concentration (R2 = 0.92, p < 0.01) and the PN/PS ratio (R2 = 0.84, p < 0.01) in the loosely bound EPS (LB-EPS) fraction. Meanwhile, the average MW of DOM in the LB-EPS and tightly bound EPS (TB-EPS) fractions also had a good correlation with the normalized CST (R2 > 0.66, p < 0.01). According to EEM fluorescence spectroscopy, tryptophan-like substances intensities in the slime, LB-EPS and TB-EPS fractions were correlated with the normalized CST. The organic matters in the EPS matrix played an important role in influencing sludge dewaterability.