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Abstract Long-term live cell tracking is desirable and necessary to understand the dynamics and complexity of biological interactions in stem cells and cancer cells. Conventional live cells fluorescence trackers are generally non-degradable and are showing increased toxicity concerns during the long-term application. Previously we developed biodegradable fluorescent poly(citrate)-based hybrid elastomers for bone regeneration applications. Here, we fabricated the photoluminescent poly(citrate-siloxane) nanoparticles (PCSNPs) through an oil/water emulsion method and demonstrated their long-term live stem cells/cancer cells imaging applications. PCSNPs showed a uniform size distribution (mean diameter 120 nm) and highly stable dispersability (above 30 days) in various physiological medium, as well as excellent fluorescent properties and photostability. PCSNPs possess excellent cellular biocompatibility, which could be efficiently internalized by cells and selectively image the cell lysosome with a high photostability. Compared with commercial Cell Tracker™ Green and Cell Tracker™ Red, the adipose-derived mesenchymal stem cells or human hepatoma cells were stably labeled by PCSNPs for over 14 days as they grew and developed (7 passages). Additionally, PCSNPs efficiently tracked cells up to 7 days in vivo through a non-invasively way compared with 1 day of commercial tracker. This study demonstrates an important strategy to design biodegradable multifunctional delivery platforms for biomedical applications such as long-term bioimaging. Graphical abstract Unlabelled Image Highlights • Biodegradable PCS nanoparticles were fabricated through a facile emulsion technology. • PCSNPs showed a monodispersed size (120 nm) and highly stable dispersability. • PCSNPs exhibited excellent fluorescent properties and photostability. • PCSNPs possess excellent cellular biocompatibility and long term cell tracking. [ABSTRACT FROM AUTHOR]