Hepatic ischemia-reperfusion injury (HIRI) is a relatively common complication of liver resection and transplantation that is intimately connected to oxidative stress. The superoxide anion radical (O2•-), as the first reactive oxygen species produced by organisms, is an important marker of HIRI. The endoplasmic reticulum (ER) is an essential site for O2•- production, especially ER oxidative stress, which is closely linked to HIRI. Thus, dynamic variations in ER O2•- may accurately indicate the HIRI extent. However, there is still a lack of tools for the dynamic reversible detection of ER O2•-. Therefore, we designed and prepared an ER-targeted fluorescent reversible probe DPC for real-time tracing of O2•- fluctuations. We successfully observed a marked increase in ER O2•- levels in HIRI mice. A potential NADPH oxidase 4-ER O2•--SERCA2b-caspase 4 signaling pathway in HIRI mice was also revealed. Attractively, DPC was successfully used for precise fluorescent navigation and excision of HIRI sites.