Pyrolusite was added in the bioleaching process to enhance the bio-oxidation process. Bioleaching tests at different dosages of pyrolusite ore, pH and inoculation amounts of Acidithiobacillus ferrooxidans were studied. The results showed that the time of the bio-oxidation process was decreased obviously and the arsenic leaching rate reached 94.4% after the bioleaching. The bio-oxidation of arsenopyrite and the effective extraction of manganese from pyrolusite were achieved by the bioleaching process. After bioleaching, the leaching rate of gold from the reaction residues reached 95.8% by cyanide leaching. In the bio-oxidation process, pyrolusite increased the redox potential of the solution to accelerate the bioleaching rate. The experiment showed that there were two reaction modes in the bioleaching process.