The fabrication of plasmonic nanostructures with sub-10 nm gaps supporting extremely large electric field enhancement (hot-spot) has attained great interest over the past years, especially in ultra-sensing applications. The "hot-spot" concept has been successfully implemented in surface-enhanced Raman spectroscopy (SERS) through the extensive exploitation of localized surface plasmon resonances. However, the detection of analyte molecules at ultra-low concentrations, i.e., down to the single/few molecule level, still remains an open challenge due to the poor localization of analyte molecules onto the hot-spot region. On the other hand, three-dimensional nanostructures with multiple branches have been recently introduced, demonstrating breakthrough performances in hot-spot-mediated ultra-sensitive detection. Multi-branched nanostructures support high hot-spot densities with large electromagnetic (EM) fields at the interparticle separations and sharp edges, and exhibit excellent uniformity and morphological homogeneity, thus allowing for unprecedented reproducibility in the SERS signals. 3D multi-branched nanostructures with various configurations are engineered for high hot-spot density SERS substrates, showing an enhancement factor of 10 11 with a low detection limit of 1 fM. In this view, multi-branched nanostructures assume enormous importance in analyte detection at ultra-low concentrations, where the superior hot-spot density can promote the identification of probe molecules with increased contrast and spatial resolution.