Abstract: Bioleaching is confronted with problems, such as low efficiency, long production cycle length, and vegetation destruction. In order to solve problems above, fly ash and low-grade copper sulfide ores were used to investigate bioleaching behaviors and bacterial community succession. Results showed that copper recovery, bacterial concentration, total proportion of main leaching bacteria including Acidithiobacillus ferrooxidans, Acidibacillus ferrooxidans, and Leptospirillum ferriphilum, were improved through using appropriate dosage of fly ash. The maximum copper recovery of 79.87% and bacterial concentration of 7.08 × 10⁷ cells·mL⁻¹ were obtained after using 0.8 g·L⁻¹ fly ash. Exclusive precipitation including Zn(Fe₃(SO₄)₂(OH)₆)₂ and Mg(Fe₃(SO₄)₂(OH)₆)₂ was found in sample added 0.8 g·L⁻¹ fly ash, which reduced the effect of hazardous ions on bacteria and thus contributing to bacterial proliferation. Bacterial community structure was differentiated, which indicated difference between original inoculation and sample used 0.8 g·L⁻¹ fly ash was less than others. Total proportion of the three microorganism above accounted for more than 95% in all tests, especially in sample with 0.8 g·L^–1 fly ash up to 99.81%. Cl^– and Ag⁺ contained in fly ash can act as catalytic agent, which contributed to conversion from smooth and dense passivation layer to sparse and scattered one, and therefore improving contact between ores, lixiviant, and bacteria. Using appropriate dosage of fly ash showed prospects in bioleaching.