Three-dimensional (3D) nanoporous gallium nitride (PGaN) scaffolds are fabricated by Pt-assisted electroless hydrofluoric acid (HF) etching of crystalline GaN followed by in situ electroless deposition of Ag nanostructures onto the interior surfaces of the nanopores, yielding a large surface area substrate for surface-enhanced Raman scattering (SERS). The resulting 3D SERS-active substrates have been optimized by varying reaction parameters and starting material concentration, exhibiting enhanced Raman signals 10–100× more intense than either (1) sputtered Ag-coated porous GaN or (2) Ag-coated planar GaN. The increase in SERS signal is attributed to a combination of the large surface area and the inherent transparency of PGaN in the visible spectral region. Overall, Ag-decorated PGaN is a promising platform for high sensitivity SERS detection and chemical analysis, particularly for reaction and metabolic products that can be trapped inside the highly anisotropic nanoscale pores of PGaN. The potential of this sampling mode is illustrated by the ability to acquire Raman spectra of adenine down to 5 fmol. Additionally, correlated SERS and laser desorption/ionization mass spectrometry spectra can be acquired from same sample spot without further preparation, opening new possibilities for the investigation of surface-bound molecules with substantially enhanced information content. Copyright © 2012 John Wiley & Sons, Ltd.