Chronic wound healing is one of the most complicated biological processes in human life, which is also a serious challenge for human health. During the healing process, multiple biological pathways are activated, and various kinds of reactive oxygen species participate in this process. Hydrogen peroxide (HO) involves in chronic wounds and its concentration is fluctuated in different pathological stages during the wound healing process. Therefore, HO may be recognized as a powerful biomarker to indicate the wound healing process. However, the pathological roles of HO cannot be fully understood yet. Herein, we proposed a near-infrared fluorescent probe DCM-HO for highly sensitive and rapid detection of HO in living cells and scald and incision wound mice models. DCM-HO exhibited a low detection limit and high specificity with low cytotoxicity for HO, which had great potential for its application in vivo. The probe was successfully utilized to monitor the fluctuation of endogenous HO in the proliferation process of human immortalized epidermal (HACAT) cells, which confirmed that HO participated in the cells' proliferation activity through a growth factor signaling pathway. In the scald and incision wound mice models, HO concentration fluctuations at different pathological stages during the wound healing process could be obtained by fluorescence imaging. Finally, HO concentrations in different stages of human diabetic foot tissues were also confirmed by the proposed probe. We expect that HO could be a sensitive biomarker to indicate the wound healing process.