Much work has been done on collapsed chains of conjugated semiconducting polymers and their applications as fluorescent probes or sensors. On surfaces spin-coated with semiconducting polymers, excitation energy transfer along the polymer backbone can be used to quickly and efficiently funnel energy to chromophores with localized energy minima. If each chromophore is immobilized within its matrix, this can result in a large fluorescence anisotropy. Through nanoprecipitation of a matrix polymer blended at low mass ratios with short-chain, hydrophobic, fluorescent semiconducting polymers, we took advantage of this large fluorescence anisotropy to make polarization-sensitive nanoparticles (NPs). These NPs are small (∼7 nm in diameter), exhibit a high quantum yield of 0.75, and are easily functionalized to bind to protein targets. Excitation of the NPs with polarized light on a wide-field fluorescence microscope enabled monitoring of both protein location and changes in protein orientation.