The Shitouzui Cu-Fe deposit is one of the typical skarn deposits in the southeastern Hubei Province. Currently, its mineralization age and the evolution process of ore-forming fluids are not clear yet. Based on the field research and petrographic observation, three generations of garnets were identified in this deposit, and in situ EMPA, LA-ICP-MS trace element and U-Pb dating of garnets were conducted for a systematic study. The results indicate that Grt1 (Adr_61~68Grs_28~36) has small compositional variation, and formed by diffusion metasomatism; Grt2 (Adr_63~89Grs_7~33) with oscillatory zoning exhibits significant compositional variation and formed by infiltration metasomatism; the composition of Grt3 (Adr_82~89Grs_5~11) is uniform and it resulted from crystallization of residual fluid. Y/Ho values (19.24~32.13, averaging at 26.46) suggest that the garnets in the Shitouzui deposit have the same magmatic fluid source. Relatively LREE-enriched and HREE-depleted patterns reflect that all these garnets precipitated in weakly acidic fluid condition. The characteristics of δEu, Sn and REE concentration indicate that the ore-forming fluid changed from relative reduced to oxidized condition, the intensity of water/rock reaction showed an increase to decrease feature, respectively, during the formation from Grt1, Grt2 to Grt3. The LA-ICP-MS U-Pb dating result of Grt2, 138 ±1 Ma, accurately constrains the Cu-Fe mineralization age of the Shitouzui deposit and further confirms the close genetic relationship between the mineralization and the Tonglüshan quartz diorite. The mineralization of the Shitouzui Cu-Fe deposit is a response to the early Yanshanian magmatic activity in southeastern Hubei Province.