A process of biooxidation followed by thiosulfate leaching of gold from refractory gold concentrate was investigated. Mineralogical studies on the concentrate showed that very fine gold grains (<10 µm) were encapsulated in pyrite and arsenopyrite, while the proportion of monomer gold was only 21%. The gold-bearing sample was identified as a high-sulfur fine-sized wrapped-type refractory gold concentrate. The gold leaching efficiency obtained by direct cyanidation was only 59.86%. After biooxidation pretreatment, the sulfide minerals were almost completely decomposed, 92wt% of the mineral particles of the biooxidation residue were decreased to <38 µm, and the proportion of monomer gold in the biooxidation residue was over 86%. Meanwhile, the gold content in the biooxidation residue was enriched to 55.60 g/t, and the S, Fe, and As contents were reduced to approximately 19.8wt%, 6.97wt%, and 0.13wt%, respectively. Ammoniacal thiosulfate was used for gold extraction from the biooxidation residue of the refractory gold concentrate. The results showed that the optimal reagent conditions were 0.18 M thiosulfate, 0.02 M copper(II), 1.0 M ammonia, and 0.24 M sulfite. Under these conditions, a maximum gold leaching efficiency of 85.05% was obtained.