Flume experiments were conducted to investigate the distribution and composition of extracellular polymeric substances (EPS) in biofilms and the detachment mechanism of biofilms grown under different flow velocity conditions. The results of biofilm growth kinetics showed that the growth trends were coincident with the logistic growth model. The growth kinetics parameters reached their maximum under intermediate velocity (IV) condition. Biofilms exhibited different profiles of EPS composition and distribution, depending on the flow conditions in which the biofilms were grown. The amounts of total polysaccharide and total protein in the thin biofilm (high velocity condition 2 – HV2) were both generally greater than those in the thick biofilm (IV). As compared to the heterogeneous distribution of EPS in the thick biofilms (IV), the EPS in the thin biofilms (HV2) exhibited more homogeneous distribution, and the bound EPS in the thin biofilms (HV2) were much greater than those in the thick biofilms (IV). From the detachment tests, an inverse relationship was observed between the proportion of detached biomass and the value of flow velocity during growth. Biofilms grown under higher velocities showed stronger cohesion than those grown under lower velocities. Therefore, water velocity during biofilm growth conditioned the distribution and composition of EPS, as well as its detachment characteristics under higher shear stress.