We report the preparation of fluorescent nanospheres based on conjugated polymers, which enables a facile fluorescence color tuning. The fluorescent nanospheres have aldehyde groups on the surface that enable the introduction of a protein ligand, biotin. The intrinsic fluorescence of the nanospheres allows detection of a dye-labeled target protein, streptavidin, via Förster resonance energy transfer. The controlled biofunctionalization of conjugated polymer-based fluorescent nanospheres represents a novel approach with high applicability to sensing of biological molecules.