Abstract: The effects of pH value on bio-oxidation of gold-containing pyrite by moderately thermophiles were studied. During the bio-oxidation process of gold-containing pyrite, the acid produced from oxidation of gold-containing pyrite leads to the decreased biological oxidation system. After stirring oxide for 5 days, the pH value was dropped to around 0.9, which was lower than the most favorable pH of moderately thermophiles. Therefore, the microbial growth and biological oxidation of gold-containing pyrite were refrained. The concentration of iron ions was increased to 15.75 g/L and 13.58 g/L respectively when the pH values of the bio-oxidation system was controlled to 1.2 and 1.5, which greatly promoted the bio-oxidation of gold-containing pyrite. XRD analysis showed that the jarosite and elemental sulfur product without generate during the process of gold-containing pyrite oxidized by moderately thermophiles.