Three-dimensional gold nanoarchitecture was fabricated by layer-by-layer (LbL) deposition of gold nanoparticles (AuNPs) and multiwalled carbon nanotubes (MWCNTs) on a glass substrate for a highly sensitive plasmonic biosensor using a conventional UV-vis instrument. Carboxyl-functionalized MWCNTs were reacted with 3-mercaptopropyltriethoxysilane (MPTES) to introduce multiple thiol groups onto MWCNTs. A self-assembled monolayer (SAM) of AuNPs on a glass chip was sequentially dipped into MPTES-functionalized MWCNTs (MWCNT-Si-SH) and AuNPs to form multilayers of AuNPs on MWCNTs. Such three-dimensionally assembled AuNPs provided a large surface area and multiple binding sites within a few steps of modification and microporous structures of multilayered MWCNTs to allow a high accessibility of target molecules. It was shown that the bulk refractive index (RI) sensitivity of these multilayered AuNPs (three-dimensional chip) appeared to be 5.6 times better than that of a monolayer of AuNPs on a glass chip (two-dimensional chip). The three-dimensional chips were further used for a biomolecular binding study, showing a detection limit as low as 0.5 nM for streptavidin and 3.33 nM for anti-human serum albumin (HSA), both of which were approximately 20 times higher than the sensitivity of the two-dimensional chips.