AbstractThe therapeutic efficacy of natural killer (NK) cells‐based immunotherapy is greatly related with the survival of transplanted NK cells. However, no effective strategy was reported to monitor NK cell viability in adoptive immunotherapy in vivo. Herein, we develop a ratiometric NIR‐II fluorescence imaging strategy to quantitively track and visualize the adoptive NK cell viability in vivo in real‐time. The nanoprobe consists of lanthanide‐based down‐conversion nanoparticles (DCNP) coated with IR786s, a reactive oxygen species (ROS) sensitive to NIR dye, which was directly labeled with NK cells. Upon cell death, the excessive ROS generation occurred within NK cells, along with IR786s degradation, turning on NIR‐II fluorescent signal at 1550 nm of DCNP under 808‐nm excitation, while the fluorescent signal at 1550 nm of DCNP under 980‐nm excitation was stable. Such an intracellular ROS‐induced ratiometric NIR‐II fluorescent signal was validated to correlate well with NK cell viability in vivo. Using this nanoreporter, we further demonstrated that co‐treatment with IL‐2, IL‐15, and IL‐21 could improve NK cell viability in vivo, achieving enhanced immunotherapy for orthotopic hepatocellular carcinoma. Overall, this strategy allows for longitudinal and quantitative tracking of NK cell viability in NK cell‐based immunotherapy.