Two bacterial stains were isolated from the activated sludge and identified as Leucobacter sp. and Alcaligenesfaecalis by 16S rDNA sequencing. Pure cultures of these two strains, representing well or poorly settled bacteria, were used to investigate the mechanism of bioflocculation in activated sludge. Based on the analyses of the characteristics of cells hydrophobicity, ζ-potential, flocculation ability and extracellular polymeric substance (EPS) composition under different growth stages, it was found that the ratio of cell EPS protein had the highly influence on ζ-potential and hydrophobicity, which were important factors to bioflocculation. Cellulase and Proteinase K could destroy the extracellular biopolymer and resulted in a decrease in the hydrophobicity and ζ-potential. However, in our study, the flocculation characteristics exhibited differently in relation to cellulase and Proteinase K. Flocculation of cells treated with cellulase and Proteinase K decreased sharply, and then recovered quickly in cellulase treatment, while cells treated with Proteinase K showed no sign of recovery. This reveals that the presence of protein in extracellular biopolymer plays an important role to the bioflocculation of ceils.