An ultra-bright fluorescence probe comprising conjugated polymer nanoparticles is developed for biological imaging. Highly blue fluorescent polyfluorene nanoparticles (PF-NPs) stabilized by sodium dodecylsulfate with an average diameter of 100 nm are prepared by a miniemulsion technique. A folate-conjugated cationic triblock copolymer is employed to coat negatively charged PF-NPs via electrostatic interaction for specific cell imaging of folate receptor over-expressing cancer cells. The coated PF-NPs show a similar size and morphology to the pristine PF-NPs, while the fluorescence intensity is enhanced. Such surface-functionalized PF-NPs are demonstrated to be suitable probes for efficient cell imaging of folate receptor over-expressing KB cells by CLSM and flow cytometry.