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The severity of environmental pollution from acid mine drainage (AMD) is increasingly garnering attention. In this study, the effects of hydraulic shear forces (achieved by regulating the shaking table’s rotation speed) on Fe²⁺ bio-oxidation and Fe³⁺ hydrolytic mineralization in an acidic 9K medium-FeSO₄-<i>Acidithiobacillus ferrooxidans</i> system (simulated AMD) are investigated. Results reveal that a higher shaking speed favors a higher oxidation rate of Fe²⁺, whereas a very low or high shaking speed restricts the removal of Fe³⁺. Shaking table rotation speeds of 120–180 rpm were preferred for biomineralization treatment in the simulated AMD. As the initial concentration of Fe²⁺ in the system decreased from 9.67 to 0 g/L in 40 h, the dissolved O₂ (DO) in the solution dropped to its lowest concentration after 20 h and then increased to its initial level between 40 and 120 h. However, the corresponding total Fe (TFe) precipitation efficiency increased with the increasing mineralization time after 40 h. The effect of O₂ supply time on biomineralization revealed that DO was mainly used in Fe²⁺ bio-oxidation. After Fe²⁺ was completely oxidized by <i>A. ferrooxidans</i>, the precipitation efficiency of TFe was independent of the O₂ supply.