The Middle-Lower Yangtze River Metallogenic Belt (MLYMB) is an important mineral resource region in China. High-resolution crustal models can provide crucial constraints to understand the ore-forming processes and geodynamic setting in this region. Using ambient seismic noise from 107 permanent and 82 portable stations in the MLYMB and the adjacent area, we present a new high-resolution 3D S-wave velocity model of this region. We first extract 5–50 s Rayleigh wave phase velocity dispersion data by calculating ambient noise cross-correlation functions (CFs) and then use the surface wave direct inversion method to invert the mixed path travel times for the 3D S-wave velocity structure. Checkerboard tests show that the horizontal resolution of the 3D S-wave velocity model is approximately 0.5°–1.0° and that the vertical resolution decreases with increasing noise and depth. Our high-resolution 3D S-wave velocity model reveals: (1) A V-shaped high-velocity zone (HVZ) is located in the lower crust and the uppermost mantle in the study region. The western branch of the HVZ passes through the Jianghan Basin, the Qinling-Dabie orogenic belt and the Nanxiang Basin. The eastern branch, which almost completely covers the main body of the MLYMB, is located near the Tanlu Fault. The low-velocity anomalies between the western and eastern branches are located in the area of the Qinling-Dabie orogenic belt. (2) High-velocity uplifts (HVUs) are common in the crust of the MLYMB, especially in the areas near the Tanlu Fault, the Changjiang Fault and the Yangxin-Changzhou Fault. The intensities of the HVUs gradually weaken from west to east. The V-shaped HVZ in the lower crust and uppermost mantle and the HVUs in the middle and lower crust likely represent cooled mantle intrusive rocks. During the Yanshanian period, fault systems formed in the MLYMB due to the convergence between the South China Plate and the North China Plate, the multiple-direction drifting of the Paleo-Pacific Plate and its subduction beneath the Eurasian Plate. The dehydration of the cold oceanic crust led to partial melting in the upper mantle. Temperature differences caused strong convection of the upper mantle material that underplated the lower crust and rose to near the surface along the deep fault systems. After mixing with the crustal materials, mineralization processes, such as assimilation and fractional crystallization, occurred in the MLYMB.