Hyperbranched polymers have attracted wide research attention owing to their unique topological structure, physicochemical properties and great potential for applications such asadditives, drug delivery, catalysts and nanotechnology. Among these, the polyamidoamine(PAMAM) dendrimers are some of the most important dendrimers. However, the synthesis and biomedical applications of fluorescent PAMAM dendrimers have received only limited attention. In this work, we present a rather effective and convenient approach for synthesis of fluorescent PAMAM dendrimers with aggregation-induced emission (AIE) properties through a one-pot catalyst-free Mannich reaction under rather mild experimental conditions (e.g., low reaction temperature, air atmosphere in the presence of water). The obtained AIE-active amphiphiles (PhE-PAD) could self-assemble into fluorescent organic nanoparticles (FONs). The obtained AIE-active FONs (PhE-PAD FONs) were fully characterized, and their successful construction was confirmed by 1H NMR spectroscopy, FT-IR spectroscopy and transmission electron microscopy. Fluorescence and UV–Visible absorption spectroscopy results demonstrated that the final PhE-PAD FONs showed strong yellow fluorescence, desirable photostability and good water dispersity. The cell viability evaluation and confocal laser scanning microscope imaging results suggested that PhE-PAD FONs possessed low cytotoxicity and excellent biocompatibility. Taken together, these results demonstrate that we have developed a facile and efficient strategy for the fabrication of AIE-active FONs, which possess many desirable features for biomedical applications.