Afterglow　fluorescence　imaging　has　been　extensively　assessed　in　ultrasensitive　bioimaging.　Since　it　eliminates　the　need　for　real-time　excitation　light　and　thereby　circumvents　the　autofluorescence　background　of　tissue,　it　holds　tremendous　potential　in　accurate　biomedical　imaging.　However,　current　afterglow　probes　are　rare　and　emit　light　only　in　the　visible　to　near-infrared　(NIR)　range,　which　is　inadequate　for　in　vivo　imaging.　To　resolve　this　issue,　an　ultrasound　(US)-activated　NIR-II　afterglow　luminescence　probe　(NPs-Ce4-SN)　emitting　afterglow　luminescence　with　a　peak　at　∼1100　nm　was　developed.　This　peak　is　nearly　400　nm　red-shifted　compared　with　other　reported　afterglow　probes.　Of　note,　after　US　termination,　NPs-Ce4-SN　undergoes　energy　transformation　to　produce　1O2　and　subsequently　undergoes　internal　oxidation-reduction　reaction　to　produce　NIR-II　afterglow,　generating　high　signal-to-noise　ratio　and　high-penetration　depth　imaging.　In　vitro　and　in　vivo　NIR-II　afterglow　imaging　experiments　revealed　that　NPs-Ce4-SN　has　good　biocompatibility　and　deep　tissue　penetration　depth,　suggesting　a　diagnostic　strategy　for　in　vivo　tumor　imaging　with　a　high　signal-to-noise　ratio.　©　2024　American　Chemical　Society.