The specific binding of fluorescent probes or biomolecules to the actin cytoskeleton network is increasingly important for monitoring various complex cellular activities such as cell adhesion, proliferation, locomotion, endocytosis, and cell division. However, improving cell uptake and subcellular resolution is still the main obstacle for successful and wide application of cellular fluorescent probes. Here, we designed and synthesized an amphiphilic block polyurethane with peculiar photophysical properties of aggregation induced emission (AIE), which can be used in living cell imaging to promote selective visualization of cell structures. The AIE effect polyurethane (abbreviated as AIE-PU) was prepared by two-step polymerization of diisocyanate terminated polyethylene glycol and polycaprolactone with hydroxyl terminated AIE dye. A series of characterization techniques proved the successful synthesis of AIE-PU. Due to the amphiphilic chain segment of its linear block molecule, AIE-PU block copolymers can self-assemble into spherical nanoparticles in aqueous solution, showing relatively stable photophysical properties and good water dispersion. Cellular experiments demonstrated that AIE-PUs have low toxicity and high actin network affinity. Moreover, the uptake mechanism was studied by low temperature and metabolic inhibition experiments, showing that AIE-PU nanoparticles could be easily internalized into different living cells through energy-dependent endocytosis, and can be transported from the cellular periphery to the actin network via clathrinand caveolae-dependent transport pathway. Upon binding with the actin network, the inter-chain AIE mechanism of the probe was significantly enhanced, which is pivotal for the long-term stable fluorescence imaging of actin microfilament network in living cells. Finally, compared with commercial actin dyes, this probe showed higher photostability, even after a longer retention time, without significant fluorescence quenching. (C) 2020 Elsevier Inc. All rights reserved.