In this work, we demonstrate a convenient and robust strategy for efficient fabrication of high fluorescence quantum yield (QY, 49.8 ± 3%) semiconducting polymer nanoparticles (SPNs), with size comparable with semiconductor quantum dots (Qdots). The SPNs were synthesized by co-precipitation of hydrophobic semiconducting polymer together with amphiphilic multidentate polymer. Comprehensive spectroscopic and microscopic characterizations showed that the SPNs possess superior photophysical performance, with excellent fluorescence brightness and reduced photoblinking in contrast with Qdots, as well as good photostability compared to a fluorescent protein of a similar size, phycoerythrin. More importantly, by conjugating membrane biomarkers onto the surface of SPNs, it was found that they were not only suitable for specific cellular labelling but also for single particle tracking because of the improved optical performance.