The Yong’an-Meixian Late Paleozoic Hercynian depression, located in western Fujian-eastern Guangdong, is an important metallogenic belt in China. The Yushui copper-polymetallic massive sulfide deposit from the middle part of the depression, with extremely high copper grades, has attracted considerable attention and research interest from geologists for years. In most previous research, the ore-forming material source and metallogenic process were inferred from indirect evidence (i.e., using H-O-C-S isotopic systematics as geochemical tracers). In this paper, the ore-forming process of the Yushui deposit has been studied directly by using nontraditional stable (Fe–Cu) isotopes for the first time, providing new evidence for the genesis of this deposit. The results show that there is a relatively negative Fe-Cu isotopic composition in the Yushui deposit, with δ56Fe values ranging from −0.519 to −1.063‰ and δ65Cu values ranging from −1.539 to −1.609‰, respectively. The fractionation of Fe isotopes is primarily attributed to hydrothermal leaching of the basement strata by ore-forming fluids, along with rapid precipitation of sulfides during the ore-forming process. On the other hand, the fractionation of Cu isotopes is probably controlled by the relatively low temperature of ore formation, sulfide precipitation and the involvement of organic matter in mineralization. Combining our findings with previous studies, the ore-forming materials of the Yushui deposit are likely derived from the basement ore-bearing strata (pre-Devonian strata) through leaching by hydrothermal fluids. Moreover, some of the ore bodies might have been locally overprinted by late-stage hydrothermal reworking and alteration.