Toxic hepatitis which is induced by chemical substance is a serious threat to human health. More and more studies have shown that peroxynitrite (ONOO−) is related with the development of toxic hepatitis. So it is important to find a tool to study ONOO− change during the diagnosis and therapy of toxic hepatitis. Herein, a series of novel near-infrared (NIR) fluorescence dyes (DDM-R) with long emission wavelength (740–770 nm) and large Stokes shift (~200 nm) are developed. Among the dyes, DDM-OH with great spectral performance and facilely modified feature is used to construct probe DDM-ONOO-. The probe have the preference of high sensitivity and excellent selectivity for ONOO−. In addition, DDM-ONOO- was applied in detecting exogenous and endogenous ONOO− in cells and further used in detecting ONOO− of CCl4-induced toxic hepatitis in cells by fluorescence imaging, 3D quantification analysis, flow cytometry. More importantly, by visualizing ONOO−, the probe was used to monitor the diagnosis of CCl4-induced toxic hepatitis in mice and evaluate the therapeutic efficacy of hepatoprotective medicines (NAC, SM, DDB). The results show that the probe will provide a powerful tool for the diagnosis and treatment of toxic hepatitis.