There have been intense efforts to stack 2D planar nanostructures in order to amplify its photonic functionality or acquire photonic chirality. In this study, we fabricated multi-layer structures layer-by-layer via consecutive execution of e-beam lithography (EBL) and 3D assembly of nanoparticles. Each layer of multi-layer 3D nanostructures is fabricated through 3-step procedures. (1) Patterns on silicon substrate via EBL. (2) Ion assisted aerosol lithography (IAAL) to fabricate 3D nanostructures. (3) Sintering 3D nanostructures by e-beam irradiation to endure the spin-coating of e-beam resistor (ER). Multi-layer 3D nanostructures were manufactured through the repetition of (1) to (3). Then, finally, ER was eradicated through O2 plasma ashing. In addition, after fabricating the multi-layer 3D nanostructures, we compared the surface enhanced Raman spectroscopy (SERS) signals of 3D mono-layer structures with the signals of 3D multi-layer structures. As a result, it was shown that the peak signal intensity of the SERS of the bi-layer nanostructure was twice as high as the monolayer’s.