Plasmonic biosensors have demonstrated superior performance in detecting various biomolecules with high sensitivity through simple assays. Scaled-up, reproducible chip production with a high density of hotspots in a large area has been technically challenging, limiting the commercialization and clinical translation of these biosensors. A new fabrication method for 3D plasmonic nanostructures with a high density, large volume of hotspots and therefore inherently improved detection capabilities is developed. Specifically, Au nanoparticle-spiked Au nanopillar arrays are prepared by utilizing enhanced surface diffusion of adsorbed Au atoms on a slippery Au nanopillar arrays through a simple vacuum process. This process enables the direct formation of a high density of spherical Au nanoparticles on the 1 nm-thick dielectric coated Au nanopillar arrays without high-temperature annealing, which results in multiple plasmonic coupling, and thereby large effective volume of hotspots in 3D spaces. The plasmonic nanostructures show signal enhancements over 8.3 × 10 8 -fold for surface-enhanced Raman spectroscopy and over 2.7 × 10 2 -fold for plasmon-enhanced fluorescence. The 3D plasmonic chip is used to detect avian influenza-associated antibodies at 100 times higher sensitivity compared with unstructured Au substrates for plasmon-enhanced fluorescence detection. Such a simple and scalable fabrication of highly sensitive 3D plasmonic nanostructures provides new opportunities to broaden plasmon-enhanced sensing applications.so-called "plasmonic hotspots" are the fundamental basis of numerous promising technologies in the fields of plasmonenhanced spectroscopy, [1][2][3][4][5][6][7][8][9][10] plasmonic biosensing, [11][12][13][14] photocatalysis, [15][16][17][18][19] and nanophotonics. [20,21] One major challenge in expanding the use of plasmonenhanced applications lies in reproducible fabrication of high-density plasmonic hotspots over large areas in a low-cost, highthroughput manner. Various methods have been explored, including aggregations of metallic nanoparticles, nanolithographic patterning, thin-film processing, and hybrid nanostructures. Despite extensive efforts, the development of a reproducible, commercially ready fabrication method that achieves both high quality (i.e., high sensitivity and good reproducibility) and high throughput (i.e., lowcost and wafer-scale fabrication) remains elusive.Among various plasmonic configurations, a nanoparticle-on-mirror (NPOM) geometry, where a nanoparticle is separated from a plain metal film by an ultrathin dielectric spacer layer, has been reported to be a highly efficient plasmonic substrate. [1][2][3][4][5] In the 2D NPOM configuration, however, electromagnetic hotspots are formed around the nanoparticle in a limited area; the effective hotspot volume accounts for a small fraction of the total