Through adding different concentration of Cu2+ in the biofilm reactor, the relationship between extracellular polymeric substance(EPS) secreted by biofilm and efficiency of copper removal by biofilm was studied. Biofilm tolerance of Cu2+ was better than activated sludge, but a high concentration of Cu2+ rapidly poisoned the biofilm. When Cu2+2 mg·L?1, Cu2+ inhibited biofilm secretion of EPS. Concentration of 2 mg·L?1Cu2+5 mg·L?1, Cu2+ would increase the amount of EPS, while Cu2+5 mg?L?1, the biofilm system displayed serious instability. Comparing proteins/polysaccharide(PN/PS) value showed that the biofilm suffered inhibition by Cu2+. The biofilm would inhibit more extracellular polysaccharide secretion, and the biofilm resistance of Cu2+ indicated that the biofilm would secrete more extracellular protein. The amount of EPS had a good correlation with the enrichment ratio of Cu2+.