Despite the large absorption cross-section of Nd3+ dopant as a sensitizer in lanthanide doped luminescence system, the strong cross-relaxation effect of it impedes the promotion of doping concentration and thus reduces the utilization of excitation light. In this work, we introduce a highly efficient acceptor, Yb3+ ion, which can quickly receive energy from Nd3+ ions, to construct an energy transfer highway for the enhancement of near-infrared emission. By using the energy transfer highway, the doping amount of Nd3+ ions in our NaYF4:Yb,Nd@CaF2 core/shell nanoparticles (CSNPs) can be markedly elevated to 60%. The quantum yield of CSNPs was determined to be 20.7%, which provides strong near-infrared luminescence for further bioimaging application. Remarkably, deep tissue penetration depth (∼10 mm) in in vitro imaging and high spatial resolution of blood vessel (∼0.19 mm) in in vivo imaging were detected clearly with weak autofluorescence, demonstrating that probes can be used as excellent NIR biosensors.