Large-scale three-dimensional(3D)hierarchical Ag nanosheet-assembled films on the indium tin oxide(ITO)glass substrates were fabricated via two-step electrochemical deposition method, and its surface-enhanced Raman scattering effect(SERS)was studied. As a large number of hot spots were located at the edges of Ag nanosheets and the cross-linking gaps between the neighboring nanosheets across the whole substrate, using 3,3′-diethylthiatricarbocyanine iodide(DTTCI)as probing molecules, the Ag nanosheet-assembled film exhibited strong SERS effect. The SERS experiments showed that the Ag film assembled by nanosheets with high pack density exhibited excellent detecting performance, which could be used as effective low-cost SERS substrate for ultrasensitive detecting of substances.