We controlled the fluorescence resonance energy transfer (FRET) between ZnO nanoparticles and rhodamine B (RB) within multilayered thin films prepared by the layer-by-layer (LbL) assembling method. Positively charged ZnO nanoparticles and RB-labeled poly(allyamine hydrochloride) (RB-PAH) were accurately incorporated into LbL assemblies of polyelectrolytes. The distance between ZnO nanoparticles and RB-PAH was adjusted by varying the number of layers of pure polyelectrolytes, leading to the controlled FRET from ZnO nanoparticles to RB-PAH.