The extracellular polymeric substances(EPS) of Acidithiobacillus ferrooxidans ATCC 23270,and iron and copper enclosed in EPS were extracted by ultrasonication and centrifugation methods to determine the interaction mechanism of Cu2+,Fe3+ and EPS during bioleaching chalcopyrite.Generally,Cu2+ ions can stimulate bacteria to produce more EPS than Fe3+ ions.The mass ratio of Fe3+/Cu2+ enclosed in EPS decreased gradually from about 4:1 to about 2:1 when the concentration of Cu2+ ions increased from 0.01 to 0.04 mol/L.The amount of iron and copper bound together by EPS in ferrous-free 9K medium containing 1% chalcopyrite was about 2 times of that in 9K medium containing 0.04 mol/L Cu2+ ions.It was inferred that the EPS with jarosites on the surface of chalcopyrite gradually acted as a weak diffusion barrier for Cu2+,Fe3+ ions transference during bioleaching chalcopyrite.