<p>Light-excited dual-mode imaging that integrates multispectral optoacoustic tomography (MSOT) and near-infrared region II fluorescence (NIR-II FL) imaging allows complementary deep-tissue visualization with high anatomical resolution and molecular sensitivity, thereby enhancing the accuracy of biomedical diagnostics. Activatable probes further advance this approach by producing signals upon recognition of disease-related biomarkers, which reduces background interference and improves imaging specificity. BTPE-NO₂@F127, a benzothiadiazole-based nanoprobe, exhibits selective activation of both optoacoustic and NIR-II FL signals in response to hydrogen peroxide (H₂O₂), a crucial early stage biomarker of liver injury, thus permitting cross-validated detection of hepatic damage in vivo with excellent signal-to-background ratio contrast. Here we present detailed procedures for preparation of the BTPE-NO₂@F127 nanoprobe and its applications in MSOT/NIR-II FL dual-mode imaging of trazodone- or ischemia–reperfusion-induced liver injury in mice. Compared to conventional liver injury diagnostic methods, such as invasive tissue biopsy, ex vivo blood analysis and previously reported fluorescent/optoacoustic probes, BTPE-NO₂@F127 offers real-time, in situ monitoring with high sensitivity and signal-to-background ratio, as well as mutually corroborating signals for increased reliability. The fabrication of BTPE-NO₂@F127, including the chemical synthesis and characterization, requires ~17 d, while the in vitro validation of its H₂O₂ responsiveness takes ~5 d. Notably, the complete workflow of data acquisition and analysis for MSOT/NIR-II FL dual-mode imaging of liver injury in mice using BTPE-NO₂@F127 can be accomplished within 10 h. The protocol is easy to follow and suitable for clinicians and researchers with a basic understanding of chemistry and bioimaging techniques.</p>