In order to investigate the enrichment of ferric iron bound by extracellular polymeric substance (EPS) on the mineral surface during bioleaching of chalcopyrite, several methods including sonication, heating and vortexing were used and sonication at 48℃ was shown as a good way to extract ferric iron. Scanning electron microscope (SEM) and energy dispersive X-ray spectrometer (EDX) analysis showed that lots of cracks and pits can be found on the chalcopyrite surface after bioleaching and that iron oxide was filled in these cracks and pits. The variations of contents of ferric iron and EPS on the chalcopyrite surface were investigated. The results indicated that the content of EPS increased rapidly in the first 10 d and then maintained at a stable level, while ferric iron content increased all the time, especially in the later stage of bioleaching.