The Tuwaishan gold deposit is located at the northeastern end of the Gezhen shear zone in the western part of Hainan Island, South China. It is one of a series of similar gold deposits hosted in the Mesoproterozoic basement rocks and structurally controlled by the Gezhen shear zone. The hydrothermal ore-forming period can be divided into quartz-pyrite-arsenopyrite stage, quartz-pyrite-base metal sulfides stage and quartz-carbonate stage. Eleven gold-bearing quartz vein samples yield δDV-SMOW and δ18OV-SMOW values of −75.9‰ to −54.4‰ and +8.1‰ to +13.7‰, respectively, and the corresponding δ18Owater values range from +3.1‰ to +8.7‰. In addition, the pyrite separates from 14 ore samples yield δ34S values of +4.5‰ to +7.9‰. The H-O-S isotopic data, along with fluid properties of the Tuwaishan and other gold deposits along the Gezhen shear zone, suggest that the ore-forming fluid and materials are of metamorphic rather than magmatic origin. Hence, we propose that the Tuwaishan gold deposit is best classified as orogenic gold deposit that resulted from regional metamorphism. Considering that the Mesoproterozoic basement rocks have experienced amphibolite facies metamorphism prior to the gold mineralization, the metamorphic devolatilization of the Ordovician-Silurian rocks at depth would provide a realistic source of fluid, gold and sulfur for the Tuwaishan and other gold deposits of the Gezhen gold belt.