Fluorophores have been extensively used as the signal mediator in biosensing and bioimaging for a long time. Enhancement of fluorescence can amplify the signal, thus improving the sensitivity, enabling earlier and accurate disease detection and diagnosis. Some metal nanoparticles, such as gold and silver, can generate a strong electromagnetic field on their surface (surface plasmon field) upon receiving photonic energy. When a fluorophore is placed in the field, the field can affect the fluorophore electrons participating in fluorescence emission and change the fluorescence output. The change can be from complete quenching to significant enhancement, depending on the metal type, particle size and shape, excitation/emission wavelengths and quantum yield of the fluorophore, and the distance between the fluorophore and the particle surface. In this study, the effects of these parameters on the fluorescence enhancement of commonly used fluorophores by gold nanoparticles (GNPs) are theoretically analyzed. Experimentally, an NIR contrast agent with enhanced fluorescence was developed by carefully tailoring the distance between Cypate (ICG based fluorophore) and a GNP, via biocompatible spacer constructs. The effect of the GNP size (3.716.4nm) and spacer length (3.24.6nm) on fluorescence enhancement was studied, and the spacer length that provided the significant enhancement was determined. The spacer of3.9nm with 16.4nm GNP provided the fluorescence of 360% of the control. The experimental data qualitatively agreed with the theoretical results and, thus, the theoretical analysis can be used as a guide for significantly improving the sensitivity of existing fluorescent contrast agents by properly utilizing GNPs and spacers.