The effect of hydraulic retention time (HRT) and pH on the biooxidation of ferrous iron during simulated acid mine drainage (AMD) treatment was investigated. The simulated AMD was highly acidic (pH 2.5), rich in iron (about 1700 mg/L) and copper (about 200 mg/L), and contained high concentrations of sulfate (about 4700 mg/L). The biooxidation of ferrous iron was studied in a laboratory-scale upflow packed bed bioreactor (PBR). The HRT was shortened stepwise from 40 h to 20 h, 13 h, and 8 h under the acidic environment at a pH value of 2.2. Then, the influent pH value was changed from 2.2 to 1.2 at a constant suitable HRT. Physiochemical and microbial community structure analyses were performed on water samples and stuffing collected from the bioreactor under different conditions. The results indicate that the efficiency of ferrous iron oxidation gradually decreased with the decrease of HRT, and when the HRT exceeded 13 h, ferrous iron in AMD was almost completely oxidized. In addition, the best efficiency of ferrous iron oxidation was achieved at the influent pH value of 1.8. Microbial community structure analyses show that Leptospirillum is the predominant genus attached in the bioreactor, and low influent pH values are suitable for the growth of Leptospirillum.