Chalcopyrite is the richest copper sulfide mineral in the world, but it is also the most resistant to biohydrometallurgical processing. To promote the bioleaching of chalcopyrite, a nonionic surfactant, t-octyl phenoxy polyethoxy ethanol (Triton X-100), was employed in this paper. Action of Triton X-100 in chalcopyrite leaching using Acidithiobacillus ferrooxidans was explored in shake flasks. Results showed that 30 mg·L⁻¹ of Triton X-100 increased the bioleaching yield of copper by 42.21% compared to the process without additive after 24 days. Under the stress of Triton X-100, the bioleaching efficiency of chalcopyrite slightly dropped at an early stage, but remarkably increased afterwards. XRD and XPS analysis of the leach residues demonstrated that potassium jarosite and elemental sulfur resulted in surface leaching passivation. Surfactant Triton X-100 appeared to induce the oxidation of elemental sulfur by bacteria owing to the increase in the sulfur surface hydrophobicity. These results suggest that Triton X-100 itself has no ability to leach chalcopyrite, but under its induction, the bioleaching of chalcopyrite can be enhanced due to the removal of the passivation layer.