Fe²⁺ bio-oxidation influenced by toxic metal ions released from the dissolution of arsenic-bearing gold ores was investigated. Fe²⁺ bio-oxidation by moderately thermophilic microorganisms was studied under different initial concentrations of Fe²⁺ and As(III), and Monod equation was used to fit the Fe²⁺ bio-oxidation under different conditions. Results showed that the Fe²⁺ bio-oxidation rate increased as the initial Fe²⁺ concentration increased until it reached 12 g/L. As(III) severely inhibited Fe²⁺ bio-oxidation. When the As(III) concentration was 8 g/L, 9 g/L Fe²⁺ was more than 200 h. The Monod equation fitted the Fe²⁺ bio-oxidation well. In the absence of As(III), the maximum specific growth rate of the culture and the substrate affinity constant were 0.142 h^-1 and 0.053 g/L, respectively. As(III) inhibited Fe²⁺ bio-oxidation via competitive inhibition, and the inhibition constant was 0.0035 g/L.