The　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.　©　2021　Wiley-VCH　GmbH