Hepatic ischemia-reperfusion injury (HIRI) is responsible for postoperative liver dysfunction and liver failure. Precise and rapid navigation of HIRI lesions is critical for early warning and timely development of pretreatment plans. Available methods for assaying liver injury fail to provide the exact location of lesions in real time intraoperatively. HIRI is intimately associated with oxidative stress which impairs lysosomal degradative function, leading to significant changes in lysosomal viscosity. Therefore, lysosomal viscosity is a potential biomarker for the precise targeting of HIRI. Hence, we developed a viscosity-activatable second near-infrared window fluorescent probe (NP-V) for the detection of lysosomal viscosity in hepatocytes and mice during HIRI. A reactive oxygen species-malondialdehyde-cathepsin B signaling pathway during HIRI was established. We further conducted high signal-to-background ratio NIR-II fluorescence imaging of HIRI mice. The contour and boundary of liver lesions were delineated, and as such the precise intraoperative resection of the lesion area was implemented. This research demonstrates the potential of NP-V as a dual-functional probe for the elucidation of HIRI pathogenesis and the direct navigation of HIRI lesions in clinical applications.