Traditional　responsive　fluorescent　probes　are　predominantly　restricted　to　qualitative　biomarker　detection,　incapable　of　delivering　real-time　quantitative　analysis　or　spatial　mapping　of　protease　activity　in　vivo,　which　is　essential　for　elucidating　disease　progression.　To　overcome　this,　a　ratiometric　second　near-infrared　region　(NIR-II)　fluorescent　(FL)　probe　(DCNP@IR-806)　was　developed　by　conjugating　caspase-3-specific　peptide　substrates　and　sensitizer　molecules　(IR-806)　to　lanthanide-doped　down-conversion　nanoparticles　(DCNP).　DCNP@IR-806　achieves　single-channel　emission　at　1550 nm　under　dual　excitation,　facilitating　self-calibrated　quantification　and　real-time　monitoring　of　activated　caspase-3　in　vivo.　Radiotherapy　induces　tumor　cell　apoptosis,　thereby　activating　caspase-3,　which　subsequently　triggers　a　ratiometric　NIR-II　FL　signal　change　of　DCNP@IR-806.　The　ratiometric　signal　demonstrates　a　linear　correlation　with　caspase-3　concentration,　achieving　a　detection　limit　of　9.96　U mL−1.　Then,　an　early　efficacy　assessment　system　capable　of　predicting　radiotherapy　outcomes　within　12　h　post-treatment　was　constructed,　markedly　expediting　evaluation　compared　to　traditional　methods　that　require　weeks.　This　rapid,　precise,　and　user-friendly　assessment　facilitates　timely　optimization　of　therapeutic　regimens　to　enhance　efficacy　while　minimizing　side　effects.　This　platform　represents　a　significant　advancement　in　precision　oncology　by　transitioning　from　qualitative　imaging　to　in　situ　quantitative　biomarker　tracking.　©　2025　Wiley-VCH　GmbH.