<p>Peroxynitrite (ONOO^-), as a crucial bioactive molecule, is closely associated with various diseases when present at abnormal levels, making reliable and accurate detection urgently needed. This study developed a colorimetric and fluorometric multi-mode sensing platform based on manganese dioxide nanosheets (MnO₂ NSs) and carbon dots (CDs) for the quantitative detection of ONOO^-. Under acidic conditions, MnO₂ NSs oxidized 3,3’,5,5’-tetramethylbenzidine (TMB) to a blue oxidized product (TMB⁺), which was subsequently further oxidized by ONOO^- to the diimine form (oxTMB). The resulting oxTMB effectively quenched the fluorescence of CDs via a fluorescence resonance energy transfer (FRET) mechanism. Additionally, ONOO^- can directly quench the fluorescence of CDs through a FRET effect. The developed multi-mode sensing strategy exhibited excellent sensitivity for ONOO^- detection. For the colorimetric mode based on TMB + MnO₂ NSs, the detection limit (LOD) was 28.73 nM in the range 1–70 µM. In the fluorescence mode based on TMB + MnO₂ NSs + CDs, the LOD was 40.17 nM in the range 0.3–45 µM. The LOD of the fluorescence mode based on CDs was 25.47 nM in the range 0.3–55 µM. By integrating a portable smartphone with a color recognition application, visual and on-site detection of ONOO^- was achieved. The proposed method was successfully applied to detect ONOO^- in human serum samples, yielding satisfactory recoveries ranging from 95.5% to 104.7%. Furthermore, CDs were successfully utilized for fluorescence imaging of ONOO^- in living cells. This multimodal sensing platform overcomes the limitations of single-mode detection and provided an economical, efficient, and convenient method for ONOO^- detection.</p> Graphical Abstract <p></p>