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Herein, we rationally fabricated three-dimensional upconversion core–double shell nanodendrites as efficient and safe luminescent probes for in vitro and in vivo bioimaging., Engineering the morphology of rare-earth doped NaYF4-based upconversion nanoparticles (UCNPs) can effectively tune their upconversion luminescence emission (UCLE) properties. Herein, we rationally synthesized a new class of three-dimensional upconversion core–double-shell nanodendrites (UCNDs) including an active core (NaYF4:Yb,Er,Ca) capped by a transition layer (NaYF4:Yb,Ca) and an active outer shell (NaNdF4:Yb,Ca). The high concentration of the Nd3+ sensitizer in the outer dendritic shell enhances the luminescence intensity, while the transition layer enriched with Yb3+ acts as an efficient energy migration network between the outer shell and inner core along with preventing the undesired quenching effects resulting from Nd3+. These unique structural and compositional merits enhanced the UCLE of UCNDs by 5 and 15 times relative to NaYF4:Yb,Er,Ca@NaYF4:Yb,Ca truncated core–shell UCNPs and NaYF4:Yb,Er,Ca spherical core UCNPs, respectively, under excitation at 980 nm. The SiO2–COOH layer coated UCNDs (UCND@SiO2–COOH) were successfully used as efficient long-term luminescent probes for in vitro and in vivo bioimaging without any significant toxicity. The uptake and retention of UCND@SiO2–COOH were mostly found in the liver and spleen. This study may open the way towards the preparation of three-dimensional UCND nanostructures for biomedical applications.