The design of tumor-targeting, intracellular protease-activatable near-infrared fluorescence (NIRF) nanoprobes is broadly interesting but remains challenging. In this work, we report the rational design of a NIR probe Cys(StBu)-Lys(Biotin)-Lys-Lys(Cy5.5)-CBT (1) to facilely prepare the self-quenched nanoparticles 1-NPs for tumor-targeted imaging in vitro and in vivo. The biotinylated 1-NPs could be actively uptaken by biotin receptor-overexpressing tumor cells via receptor-mediated endocytosis. Upon intracellular proteolytic cleavage, 1-NPs were disassembled to yield the small molecular probe Lys(Cy5.5)-Luciferin-Lys(Biotin)-Lys-OH (1-D-cleaved), accompanied by fluorescence "Turn-On". With this NIRF "Turn-On" property, 1-NPs were successfully applied for tumor-targeted imaging. We envision that our nanoparticles could be applied for fluorescence-guided tumor surgery in the near future.