Peroxynitrite (ONOO − ) is a short-term reactive biological oxidant and plays an important role in cellular signal transduction and homeostatic regulation. However, excess ONOO − is associated with neurodegenerative and cardiovascular diseases. Therefore, rapid, sensitive, and accurate assays for ONOO − detection are essential for exploring its physiological and pathological function. In this work, a wavelength-shifted and ratiometric fluorescent sensing platform for ONOO − is constructed by splitting green fluorescent carbon dots (G-CDs) and aggregating orange fluorescent carbon dots (O-CDs). The mixed G-CDs and O-CDs (M-CDs) show a fast and precise response to ONOO − in the range of 0−250 μM, with a detection limit of 10 nM. In the linearity range within 3 μM ONOO − , an obvious wavelength shift of G-CDs from 495 to 475 nm is observed owing to the oxidation and nitration of ONOO − to the surface-state fluorescence of G-CDs, accompanied by the splitting of G-CDs. In the linearity range of 3−250 μM ONOO − , the fluorescence of G-CDs remains constant, while the molecular-state fluorescence of O-CDs gradually quenches by the oxidation and nitration of ONOO − through the fluorescence static process and induces their aggregation. Additionally, M-CDs show favorable intracellular imaging of endogenous and exogenous ONOO − . This study not only presents a new fluorescence wavelength shift mechanism for ONOO − sensing but also provides insights into CDs' fluorescence mechanism by exploring their morphology and structure via reacting with reactive oxygen species (ROS).