The Mibei gold deposit, located in the southwestern part of the Xuefengshan uplift zone, the middle section of the Jiangnan orogenic belt in southern China, has estimated gold resources of approximately seven tons. This deposit is primarily a quartz vein-type gold deposit, with ore bodies occurring mainly within Neoproterozoic metasediments. The main metallic minerals in the ore are pyrite, chalcopyrite, and arsenopyrite. In this study, the petrography and microthermometry of ore-forming fluid inclusions, oxygen isotopes of gold-bearing quartz, and sulfur isotopes of gold-bearing sulfides and arsenopyrite were analyzed. Three types of fluid inclusions were identified: type Ia three-phase inclusions comprising vapor and two phases of liquids (VCO2 + LCO2 + LH2O), type Ib two-phase liquids (LCO2 + LH2O), type II two-phase vapor-rich inclusions (V/V + L > 50%), and type III pure liquid inclusions. Type I inclusions were heated uniformly to the liquid phase, type II inclusions were heated uniformly to the gas phase, and type III inclusions were heated without change. In general, the temperature range of homogenization to liquid phase of fluid inclusions in the Mibei gold deposit is 204–227℃. The salinity of the inclusion ranges from 4.6 to 12.2 wt% NaCl equiv. The δ18OSMOW of gold-bearing quartz varies from 16.9‰ to 17.5‰. The δ18OH2O of gold-bearing quartz are varied from 6.5‰ to 7.5‰. The δ34S values of gold-bearing pyrite range from 1.7‰ to 6.8‰. The δ34S values of gold-bearing arsenopyrite range from 5.6‰ to 5.9‰. The δ34S values of pyrite from wall rocks slate range from 6.4‰ to 11.6‰. This evidence implies that the ore-forming fluids of the Mibei gold deposit originated from magmatic-hydrothermal processes, mixing with minor S from the surrounding metasediments. Combined with the evolution of the Jiangnan orogenic belt, due to the magmatic and tectonic activities of the Xuefengshan uplift during the Caledonian period, the fault seal mechanism controlled the ore-forming process. Overall, the Mibei gold deposit is more akin to a magmatic-hydrothermal gold deposit.