Colorimetric detection is cost-effective and user-friendly when used for sensing target analytes without a need of bulky and expensive equipment. The extraordinary transmission phenomena through plasmonic periodic nanocup arrays achieve colorimetric sensing by detecting color changes of transmitted light associated with the refractive index variation. The application of the nanocup arrays, however, is relatively restricted due to a limited sensitivity for monolayered target analyte detections on the surface. In order to improve the sensitivity bounded by the underlying nanostructures, hybrid nanoparticle (NP)-nanocup array substrates are developed for enhancing the sensitivity to the refractive index change. The three dimensionally assembled Au NPs in circle along the sidewall of each nanocup increases the density of hot spots by the heterogeneous plasmonic coupling between the NP and the edge of the nanocup; thus a small refractive index change at the hot spot becomes easily detected than bare nanocup arrays. In addition to the bulk refractive index sensing, an ultrasensitive spectroscopic detection of the antigen-antibody binding is achieved by this three-dimensional self-assembly of Au NPs on the Au nanocup arrays.