Plasmonic fluorescent nanocomposites are difficult to prepare due to strong quenching effects on fluorophores in the vicinity of noble metal nanoparticles such as gold (AuNPs). We successfully prepared plasmonic fluorescent nanocomposites of two cyanines (1 and 2) aggregating upon 2 - 40 nm AuNPs or streptavidin-conjugated 10 nm AuNPs. We used high throughput screening (HTS) for the first time to characterize the spectral properties, aggregation kinetics, aggregation density and photostability of the nanocomposites. Fluorescence from nanocomposites declined inversely with AuNPs size: 40 nm ≥ 20 nm > 10 nm > 5 nm > 2 nm. Sensitivity (limit of detection, LOD, 10(5) - 10(11) AuNPs/mL), brightness of the nanocomposites and surface coverage of AuNPs by cyanine aggregates were all influenced by five factors: 1) AuNPs size; 2) cyanine type (1 or 2); 3) aggregate density; 4) distance between aggregates and AuNPs surface; and 5) streptavidin protein conjugation to AuNPs. We propose a model for plasmonic fluorescent nanocomposites based on these observations. Our plasmonic fluorescent nanocomposites have applications in chemical and biological assays.