Peroxynitrite (ONOO) is an extremely powerful biological oxidant and it can react with a wide variety of molecular targets to result in a series of disease states, which has made ONOO a central biological pathogenic factor. In order to disclose its various pathological events, a large number of fluorescent ONOO probes have been developed in recent years. Nevertheless, some limitations still remain, such as short excitation and emission wavelengths, long fluorescence response times, and cross-interference caused by other reactive oxygen species (ROS). In this work, a new near-infrared (NIR) fluorescent probe, SiRTA, containing an aromatic tertiary amine functional group and a Si-rhodamine fluorophore, was developed for endogenous ONOO detection and imaging. The probe exhibits not only a fast, sensitive, and specific fluorescence off-on response toward ONOO in chemical systems but also excellent imaging ability for endogenous ONOO in living cells. With the probe, the therapeutic effects of phenolic acid antioxidants in EA.hy926 endothelial cells suffering from ischemia-reperfusion injury and the pathogenesis of diabetes and diabetic nephropathy in pancreatic β-cells and diabetic rats have successfully been evaluated. These results indicate that SiRTA is a potentially outstanding imaging tool for studying the physiological and pathological events of ONOO and relevant drug therapies.