To enhance Au recovery from double refractory gold ores (DRGOs), a crucial step involves the oxidation of sulfides to release Au grains and degrading carbonaceous matter to minimize Au(CN)2− adsorption during cyanidation. Enzymatic degradation of carbonaceous matter in DRGOs has become attractive due to its environmentally friendly attributes. Laccases, the multicopper enzymes, have been extensively studied, primarily for their ability to degrade lignin, a precursor of carbonaceous matter. Various substrates oxidized by laccase can be expanded by mediators, lower-molecular-weight compounds easily oxidized by laccase, producing highly reactive cationic radicals that oxidize more complex substrates by shifting the chemical equilibrium. Various mediators have been identified, and laccases from different mushrooms exhibit distinct interactions with these mediators. Therefore, this study investigated the impact of different mediators in laccase-mediator systems on the degradation of carbonaceous matter in DRGOs to enhance Au extraction. The laccase-mediator system proposed represents a novel approach that deserves further attention, especially for understanding carbon science in hydrometallurgy.