This study explores the fluorescence enhancement of quantum dots (QDs) by gold film over nanospheres (AuFoN) plasmonic substrates, focusing on how a polymer matrix and plasmon resonances of the substrate affect the fluorescence properties of QDs. It was observed that polyvinylpyrrolidone (PVP) facilitated the uniform distribution of QDs on the surface of the AuFoN by simple drop-coating, avoiding aggregation during solvent evaporation. Progressive fluorescence redshifts and intensity enhancement were observed when moving from QDs on glass substrates to planar Au, and most pronouncedly, to nanostructured AuFoN substrates. The fluorescence enhancement was further analyzed by varying the diameter of the polystyrene spheres used in AuFoN fabrication, revealing that substrates based on 600-700 nm spheres provided the strongest fluorescence amplification due to stronger localized electromagnetic fields. Time-resolved fluorescence measurements revealed two primary fluorescence lifetime components for QDs on AuFoN: a short component linked to non-radiative plasmonic energy transfer and a long component representing intrinsic QDs emission. By optimizing sphere size, Au nanostructured films can be tailored to control QDs fluorescence lifetimes and intensity, advancing their use in biosensing, photonics, and other fluorescence-based technologies. This work enhances our understanding of how substrate design and matrix effects impact QDs fluorescence, providing a pathway for precisely engineered Surface Enhanced Fluorescence (SEF) platforms suited to various applications in optical sensing and more general photonics.