In this paper, a fluorescein methacryloyl monomer (named Flu-MA) was firstly fabricated by the esterification reaction of fluorescein and methacryloyl chloride; subsequently, a novel amphiphilic fluorescent copolymer (Flu-PEG) with smart pH sensitivity was successfully prepared by RAFT copolymerizing of Flu-MA and poly (ethylene glycol) monomethacrylate (PEGMA). The result of H-1 NMR and FTIR implied that both Flu-MA and PEGMA monomers were successfully incorporated into Flu-PEG copolymers, the molecular weight (Mn) of which was about 39,800 with polydispersity index (PDI) as about 1.30. In aqueous solution, Flu-PEG copolymers could self-assemble into fluorescent organic nanoparticles (FONs) with about 200 nm diameters due to high water dispersibility and good fluorescence, respectively, endowed by PEGMA and Flu-MA, the fluorescence intensity of which was high sensitive to pH value of solution. More importantly, due to their high water dispersibility, excellent biocompatibility and good fluorescence, the as-prepared Flu-PEG FONs were high attractive for the application in cell imaging.