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　∼20　times　higher　than　the　sensitivity　of　the　two-dimensional　chips.　©　2010　American　Chemical　Society.