The invention discloses a complex method for improving efficiency of chalcopyrite leaching conducted through sulfur oxidation cultures and belongs to the technical field of biology. The method comprises the steps that on the basis of a Starkey-chalcopyrite composite culture medium, an external-source energy substrate, namely elemental sulfur, is supplemented in a pulsed manner at the middle and later culture stages, and high-density culture of thiobacillus thiooxidans is achieved; a proper amount of elemental sulfur and iron ions are added while cell inoculation is conducted, start of a leaching biochemical reaction cycle is accelerated, the lag phase is shortened, and the inoculum size is increased; and at the later leaching stage, the low constant pH is maintained, jarosite stress is inhibited, sulfur oxidation culture cells are added as supplementation, iron metabolism is promoted while sulfur metabolism is improved, and furthermore, the leaching microenvironment is improved. By means of the method, the sulfur oxidation cultures can be cultured more efficiently, the leaching lag phase is shortened, and active biochemical reaction state of the leaching microenvironment is maintained better, and thus the leaching rate is increased; and in addition, the method is easy to operate and implement and suitable for being applied and popularized in similar bioleaching processes on a large scale.