Peroxynitrite (ONOO ) is one of the important reactive oxygen species, which plays a vital role in the physiological process of intracellular redox balance. Revealing the biological functions of ONOO will contribute to further understanding of the oxidative process of organisms. In this work, we designed and synthesized a novel red-emitting fluorescent probe MCSA for the detection of ONOO , which could rapidly respond to ONOO within 250 s and exhibited high sensitivity to ONOO with a low detection limit of 78 nM. Co-localization experiments demonstrated MCSA had the ability to localize into the mitochondria and endoplasmic reticulum. What's more, MCSA enabled monitoring ONOO level changes during tunicamycin-induced endoplasmic reticulum stress. We have also successfully achieved the visual detection of exogenous and endogenous ONOO in living cells and zebrafish. This work presented a chemical tool for imaging ONOO in vitro and in vivo.