The Huangmeijian area represents one of the major hydrothermal uranium metallogenic provinces in the middle-lower Yangtze River region. Although multiple episodes of hydrothermal overprinting have been recognized, the origin of the ore-forming fluids remains controversial. Through the study of trace element composition and lithium isotope characteristics in the industrial boreholes along the northern margin of the Huangmeijian rock mass, it was found that the lithium isotope compositions of different uranium mineralized rock types in the area exhibit significant differences. The δ⁷Li values of unmineralized host rocks show minor variations (10.3‰~ 13.2‰), while those of siliceous veins and calcite veins in the mineralized alteration zones exhibit larger discrepancies (-9.9‰~ 6.1‰). The U and Mo elements in the mineralized alteration veins and host rocks display enrichment features, whereas Sr and Ba elements are depleted. The rare earth element characteristics reveal notable enrichment of light rare earth elements (LREE), while heavy rare earth elements such as Eu, Dy, and Tm show significant depletion. These Li-isotope and trace-element characteristics indicate the involvement of at least two distinct hydrothermal fluid events. The early-stage uranium mineralization was driven by magmatic fluids exsolved from a deep lower-crustal magma chamber, whereas late-stage uranium deposition resulted from post-magmatic hydrothermal circulation. The study provides a robust example of using Li isotopes to identify and distinguish the sources of uranium ore-forming fluids, thereby greatly extending the applicability of Li isotopes in hydrothermal ore-forming systems.