We report the synthesis of star polymers with site-isolated chromophores obtained by nitroxide-mediated polymerization and reversible addition -fragmentation chain transfer techniques through the "arm first" method. Linear polymer precursors such as alpha-alkoxyamine-terminated polystyrene (PS) and thioester-terminated poly(acrylic acid) (PAA) were prepared followed by the addition of fluorescent divinyl cross-linkers derived from the fluorene and thiophene families. We first synthesized organic soluble star polymers containing hexyl-functionalized fluorene and thiophene cross-linkers with PS. Second, ethylene oxide (EO)-functionalized fluorene cross-linkers were incorporated into PAA linear precursors to give water-soluble star polymers with site-isolated chromophore core units. The photoluminescence increased significantly while the emitting wavelength corresponded with highly dilute, nonconjugated chromophores in solution. This effect is indicative for a highly localized concentration of chomophores in the star polymer cores that are covalently connected but do not show the typical effects of concentrated monomers in solution such as aggregation, fluorescence quenching, and a red shift of emission wavelength. The site isolation of chomophores in star polymers leads to nanostructures with highly photoluminescent core units while the emanating linear polymers are available for further functionalizations.