Fault blocks and fold systems distributed in Wuhu constitute an important component of the geological structural zoning in the middle-southern segment of the Tan-Lu fault zone and its adjacent areas. However, research on the distribution of low-velocity layers and the deep seismogenic background in this region remains insufficient. Using 92 986 absolute P-wave travel times and 293 165 relative travel times recorded by 101 seismic stations from 9 179 seismic events in the Wuhu area of Anhui and its vicinity from 2008 to 2020, this study obtained precise earthquake relocation results and a three-dimensional crustal velocity structure for the studied areas using the double-difference seismic tomography method. The earthquake relocation results showed that the distribution of earthquakes exhibited certain variations horizontally while becoming more clustered and regular vertically. The distribution of hypocenters was closely related to major regional faults, with fault extensions controlling the spatial distribution of earthquakes. The velocity structure characteristics at different depths in the study area displayed significant lateral heterogeneity. The Tan-Lu fault zone, as a distinct tectonic boundary, exhibited segmentation in its velocity structure: the Lower Yangtze fault block was generally characterized by low velocities, whereas the Dabie fault-fold belt and the Sulu fault block showed prominent high-velocity anomalies. The results substantially improved the accuracy of the original earthquake locations in the study area. Integrated analysis of the relocation results and vertical velocity profiles indicated a notable correlation between the mid-crustal low-velocity layer and moderate-to-strong earthquakes in the region.