Fluorescent organic nanoparticles (FONs) with aggregation-induced emission (AIE) features have been extensively investigated for bio-imaging applications because of their unique AIE properties, biocompatibility, and well designability. In this work, AIE-active FONs have been fabricated via a "one pot" mercaptoacetic acid locking imine (MALI) reaction, in which aldehyde groups containing AIE-active dye (named as CHO-An-CHO) was covalently conjugated with NH2-terminated polymers (PEG-PABA) to form amphiphilic luminescent polymers using mercaptoacetic acid (MTA) as the "lock" molecule. These final polymers (denoted as PEG-PABA-An-CHO) possessed amphiphilic properties and could self-assemble into PEG-PABA-An-CHO FONs with excellent water dispersibility. Furthermore, thus PEG-PABA-An-CHO FONs displayed enhanced luminescence in aqueous solution due to the aggregation of AIE feature of CHO-An-CHO. More importantly, thus final PEG-PABA-An-CHO FONs exhibited desirable biocompatibility and fluorescent imaging performance. As compared with traditional stepwise methods that are laborious, time-consuming and involve separation and purification of intermediate products, thus "one pot" strategy relied-on MALI reaction showed distinct advantages, including simplicity, high efficiency, universality and atom-economy. Thanks to the good applicability of MCR-MALI reaction towards different substrates, this "one pot" strategy should be a useful tool for preparing sophisticated and multifunctional AIE-active materials for different applications potential. (C) 2016 Elsevier B.V. All rights reserved.