A malignant tumor remains one of the leading causes of deaths across the world. Thus, diagnosis of tumor development with noninvasive visualizing methods is significant for tumor therapy. Herein, an activatable two-photon NIR fluorescent probe for imaging of peroxynitrite in a tumor was elaborately designed. The probe demonstrated an increased NIR emission in response to peroxynitrite , which ensured that the probe detects ONOO in cell and . Cellular imaging results disclosed that the probe was competent to detect adscititious ONOO level change in HeLa cells, as well as endogenous ONOO concentration in lipopolysaccharides (LPS) and IFN-γ-stimulated RAW 264.7 cells. Additionally, zebrafish imaging revealed that the probe accumulated in the pancreas and was lightened up by the addition of ONOO. Remarkably, the probe can be harnessed to image an ONOO production profile in xenograft 4T1 tumor mice by both one-photon and two-photon fluorescence imaging. Benefiting with the two-photon excitable properties and NIR emissive properties, the probe can be used for noninvasive imaging of ONOO in the onset and development of tumors for the first time. This work provided a noninvasive and efficient detection method for ONOO in a tumor, which would find more applications in tumor diagnosis and therapies.