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Mercury (Hg) is considered an extremely toxic heavy metal which is extremely harmful to both the human body and environment. In addition, Hg2+-induced oxidative stress also exerts a crucial role to play in pathophysiological mechanisms of mercury toxicity. Thus, efficient and specific fluorescent probes for imaging Hg2+-induced oxidative stress are necessary. In the present study, we rationally design a novel Hg2+-activated and ICT-based NIR emission fluorescent probe NIR-HOfor sequentially monitoring the ONOO–level with a “dual-key-and-lock” strategy. The probe NIR-HOshowed rapid response and excellent specificity and sensitivity for the detection of Hg2+and ONOO–in vitro. Cell imaging demonstrated that Hg2+-induced oxidative stress was involved in ONOO–upregulation. Also, GSH, NAC, and EDTA were employed as excellent detoxifying drugs against Hg2+-induced toxicity. Moreover, the probe NIR-HOwas successfully used for imaging Hg2+and ONOO–in vivo. In brief, NIR-HOprovides a simple and powerful approach which can be used to image Hg2+-induced oxidative stress in the pathological environment.