The refractory gold ores associated with rich copper and trapped in pyrite and quartz were studied. With conventional technique (all-sliming cyanidation), the gold recovery rate is only 51.78%. To eliminate the negative effects of copper and pyrite on cyanidation and increase the gold recovery rate, the investigation on bio-heap leaching pretreatment was made, by which Cu would be dissolved and gold would be liberated from pyrite. The experiment adopted mixed bacteria, mainly Thiobacillus ferrooxidan (named T. f1), as the bacterial catalyst for bio-preconditioning and was carried out in a PVC column with a diameter of 20 cm and a height of 1.3 m loaded with gold ores. The temperature was controlled between 28 and 30℃, the pH value was kept between 2.0-2.5,and the flux of sprinkling bacterial liquid was maintained 0.80 L/h. After 45-days bio-oxidization, among the samples sizing from 0to 5 mm, the oxidation rates of Cu, Fe and S were respectively 44.62%, 28.16% and 25.46%, and the gold recovery rate by cyaniding increased to 80.35%. The bio-heap leaching pretreatment can therefore effectively dissolve Cu and liberate gold from pyrite and lead to the increase of gold extraction.