A total gold extraction of 70.2% could only be reached via direct cyanidation from a refractory As-, S- and C-bearing gold concentrate calcine, and the gold extraction varied noticeably with different size fractions. The reasons for unsatisfactory gold extraction from the calcine were studied through analyses of chemical composition, chemical phase and SEM-EDS of different sizes of particles. It was found that a significant segregation of compositions occurred during the grinding of gold ore before flotation. As a result, for the calcine obtained after oxidative roasting, the encapsulation of gold by iron oxides was easily engendered in finer particles, whilst in coarser particles the gold encapsulation by silicates was inclined to occur likely due to melted silicates blocking the porosity of particles. The improvement of gold leaching from different size fractions was further investigated through pretreatments with alkali washing, acid pickling or sulfuric acid curing-water leaching. Finally, a novel process was recommended and the total gold extraction from the calcine could be increased substantially to 93.6% by the purposeful pretreatment with alkali washing for the relatively coarse size fraction (+37 μm) and sulfuric acid curing–water leaching for the fine size fraction (−37 μm).