The Mianhuakeng deposit is the most representative granite-related hydrothermal vein-type uranium deposit in South China; however, the characteristics of the mineralization-forming fluid and metallogenic mechanism are still less constrained. To address the scientific problems above, we investigated the trace element chemistry and sulfur isotope compositions in syn-mineralization pyrite and pitchblende from the Mianhuakeng uranium deposit. The trace element chemistry shows that the mineralization in the Mianhuakeng deposit belongs to an intermediate-to-low temperature hydrothermal system, which is consistent with the homogenization temperature distribution of fluid inclusions. Redox-sensitive elements (such as Co and Se) in syn-mineralization pyrite suggest a reductive nature of the mineralization-forming fluids. The fractionation between light and heavy REE in pitchblende from the Mianhuakeng deposit is most similar to those from the Changjiang pluton. The pronounced negative Eu anomaly is coincident with mineralization-bearing granites. The δ³⁴S values of syn-mineralization pyrite range from −10.2 to −1.4‰, which is higher than those values of pyrite from granites near the studied area and lower than the δ³⁴S values of pyrite from diabase in the ore district. The REE signatures of pitchblende and sulfur isotope composition of syn-mineralization pyrite suggest that the major U source for the Mianhuakeng deposit is most likely the Changjiang pluton, probably accompanied by the incorporation of mantle-derived fluids. The circulations of CO₂-rich hydrothermal fluids leached uranium from granite source rocks, especially from the Changjiang pluton. The change of physicochemical conditions of the mineralization-forming fluid resulted in the deposition of the uranium minerals in favorable structural traps to form the hydrothermal vein-type Mianhuakeng uranium deposit.