In order to improve the bio-oxidation efficiency of Axi refractory gold concentrate, a two-step process including a high temperature chemical oxidation and a subsequent bio-oxidation, combined with p H control during the bio-oxidation step was used. The results revealed that the optimum mode was to maintain solution p H at 1.0-1.2 during the biological oxidation stage. Under this condition, the activity of mixed culture could be sustained and the formation of jarosite could be diminished, thus the oxidation efficiency was improved. The oxidation levels of iron and sulfur were improved by 12.50% and 15.49%, and the gold recovery was increased by 21.02%. Therefore, the two-step process combined with p H control is an effective method for oxidizing the biohydrometallurgical process of Axi gold concentrate, and it will have a broad prospect of application in dealing with complex refractory gold concentrate.