Acidithiobacillus ferrooxidans is a chemotrophic, aerobic, acidophilic, and Gram-negative bacterium that plays a key role in iron and sulfur cycling and has a wide range of applications in the industrial field. A novel A. ferrooxidans strain, hereinafter referred to as strain “YQ-N3”, was isolated from sediments of a river polluted by acid mine drainage (AMD) of an abandoned mine in Shanxi, China. The whole genome sequencing results revealed that A. ferrooxidans YQ-N3 has a 3,217,720 bp genome, which is comprised of one circular chromosome and five circular plasmids (Plasmid A, Plasmid B, Plasmid C, Plasmid D, Plasmid E). Plasmid E, a new plasmid, had not been annotated in the reference database. A. ferrooxidans YQ-N3 had a close evolutionary relationship with A. ferrooxidans ATCC23270 and A. ferridurans JCM18981 and exhibited higher similarity in its genomic structure with A. ferrooxidans ATCC23270. Multiple genes related to environmental resistance and iron and sulfur metabolism were predicted from its genome. A. ferrooxidans YQ-N3 can remarkably increase the oxidation rate of Fe2+ and S0 and enhance the hydrophilicity of S0, which was supported by functional gene analysis and laboratory experiments. The biological desulfurization experiment demonstrated that A. ferrooxidans YQ-N3 can reduce the sulfur content in coal by removing pyrite sulfur and organic sulfur.