Thiourea is a low-toxicity gold lixiviant that has promise to replace the highly toxic cyanide for gold extraction. Chalcopyrite is an important gold-bearing sulfide mineral, and thus investigation of its influence on gold leaching is important to maximize the utilization of refractory copper sulfide gold ore. In this paper, the effect of chalcopyrite on thiourea leaching of gold and the role of oxalate were studied through a series of leaching experiments, surface characterization and DFT calculation. Results showed that the presence of chalcopyrite severely hindered thiourea leaching of gold demonstrated as the greatly decreased in gold dissolution and thiourea stability. The detrimental effect of chalcopyrite was inferred to be the catalysis of chalcopyrite on thiourea oxidation by Fe3+, leading to the much-increased thiourea consumption, and thus deteriorated the gold dissolution. Oxalate as an additive was used to improve gold dissolution and thiourea stability. Oxalate not only weakens the interaction between Fe3+ and thiourea through complexing with Fe3+, but also competes with thiourea to adsorb on the chalcopyrite surface, decreasing thiourea adsorption. Besides, the formation of Fe-oxalate complexes, as the terminal acceptor of electron, significantly reduced the oxidizability of Fe3+, thus relieving the catalysis of chalcopyrite on thiourea oxidation.