Fluorescent cross-linked nanoparticles with variable fluorophore loading amounts, locations, and particle sizes were synthesized from sequential one-pot functionalization/cross-linking of block copolymer micelles with amine-terminated dye and cross-linker molecules, via reductive amination and amidation. The fluorescence quantum yield and brightness of these nanoparticles were evaluated by steady-state and dynamic fluorescence methods. The results demonstrate that the quantum yield and brightness of the fluorescent nanoparticles correlated directly with the number of dyes/nanoparticle and the nanoparticle size. A strategy to increase the fluorescence brightness of nanoparticles with fluorescein and near-infrared dyes is proposed.