Due to the good biocompatibility, 3-polylysine (Ply) has been extensively investigated for various biomedical applications. In this study, a fluorescent monomer (named Flu-MA) was firstly synthesized through acylation reaction of fluorescein by methacryloyl chloride, and the initiator of 3-polylysine bromide (named Ply-Br) was prepared by the introduction of a bromine atom into Ply by the acylation reaction of Ply with abromoisobutyryl bromide. Subsequently, a novel amphiphilic fluorescent polymer (Flu-Ply) was successfully fabricated by ATRP via incorporation of Flu-MA monomer into Ply chains for the first time.The structure and properties of the obtained Flu-Ply fluorescent polymer were investigated in detail by 1 H NMR, TEM, UV-vis, FL and FTIR, and the results confirmed the successful incorporation of Flu-MA into Ply by ATRP. As a result of Flu-MA and Ply respectively endowing the as-prepared Flu-Ply polymer with fluorescence and water dispersibility, it tended to self-assemble into fluorescent organic nanoparticles (FONs) with excellent biocompatibility. More importantly, the good fluorescence, uniform spherical morphology, excellent biocompatibility and water dispersibility of Flu-Ply FONs exhibited an attractive prospect for bioimaging applications.