To study the influence of Acidithiobacillus ferrooxidans（At.f） mutated by X-ray on the biooxidation and bioleaching of refractory gold ores, X-ray was used to irradiate At.f strains at different doses, and more efficient strains were screened by microbiology methods and the measurement of relevant indicators. Compared with the original strain, the bioleaching ratio of iron by the mutants after irradiation at 450 Gy was 50.24%, namely 44% increased, and the bioleaching ratio of gold reached 86.68%-two folds than gold direct cyanidation leaching. It is proved that X-ray irradiation is an outstanding way to breed mutants of high bioleaching ratio of iron and gold strains of At.f.