The combination of bio-oxidation pretreatment of refractory sulfide gold ore and thiourea leaching is a suitable method for gold extraction due to its environmental friendliness and compatibility in the two leaching systems. Arsenopyrite is an important auriferous sulfide mineral where the encapsulated gold can be effectively exposed by bioleaching pretreatment. The issue of high thiourea (TU) consumption, however, severely hinders the commercial application of this technology. In this paper, the effect of arsenopyrite on gold leaching in an acidic TU solution was investigated. Leaching results showed that the presence of arsenopyrite seriously impeded TU leaching of gold represented as the much decrease in gold dissolution and TU stability. The dominant cause for the detrimental effect of arsenopyrite was that arsenopyrite can catalyze the oxidation of TU by Fe3+ and thus bring about high TU consumption, further resulting in the marked deterioration of TU dissolution of gold. The catalytic mechanism is believed to be that TU adsorbs on arsenopyrite surface and then electrons transfer from TU to the oxidant Fe3+ through the conduction bands of arsenopyrite. Oxalate was used to improve TU stability and gold dissolution, and its mechanism was proposed as follows: oxalate not only weakened the interaction between Fe3+ and TU through complexing with Fe3+ but also competed with TU to absorb on the arsenopyrite surface, which decreased the TU adsorption. Besides, the formation of Fe-oxalate complexes, as the terminal electron acceptor, distinctly reduced the oxidation capacity of Fe3+, thus alleviating the catalytic effect of arsenopyrite on TU oxidation by Fe3+.