Real-time　monitoring　the　therapeutic　process　of　sonodynamic　therapy　(SDT)　is　essential　to　optimize　the　treatment　course　in　time　and　eventually　improve　the　efficacy.　The　generation　of　singlet　oxygen　(1O2)　is　a　quintessential　characteristic　of　SDT,　which　permits　non-invasive　monitoring　of　SDT　by　real-time　imaging　of　1O2　inside　the　tumor.　Nonetheless,　the　majority　of　probes　are　unable　to　measure　1O2　in　real　time　because　of　its　short　half-life　and　strong　oxidative　capacity.　Here,　the　study　constructs　a　ratiometric　nanoplatform　(DTPI)　utilizing　two　fluorescent　probes　and　the　sonosensitizer　TiO2.　The　poisonous　1O2　generated　by　DTPI　following　ultrasonic　(US)　radiation　efficiently　destroys　tumor　cells.　The　structural　disruption　of　fluorescent　dye　IR-1061　by　1O2　leads　to　a　reduction　in　the　DTPI　fluorescence　signal　at　1100 nm,　while　US　radiation　has　no　impact　on　the　fluorescence　signal　at　1550 nm.　Thus,　DTPI　provides　a　precise　and　consistent　reflection　of　the　treatment　efficacy　at　the　tumor　site,　leveraging　the　ratiometric　fluorescence　signal　and　variations　in　oxygen　content　throughout　the　treatment　process.　This　ratiometric-fluorescence-based　reflection　strategy　establishes　an　effective　and　dependable　platform　for　the　real-time　monitoring　and　assessment　of　the　cancer　therapeutic　effect　through　ratiometric　probes.　©　2024　Wiley-VCH　GmbH.