A growing body of evidence shows that peroxynitrite (ONOO-) is involved in a large number of diseases, signal conduction, and immunoregulation in living systems. The determination of intracellular ONOO- attracts increasing attention. In this paper, a novel dicyanoisophorone-derived borate-containing near-infrared (NIR) fluorescent probe NIR-PN for sensitive detection of ONOO- was designed. Probe NIR-PN holds ultrasensitivity (DL = 5 nM), high specificity, and fast response (<3 s) for ONOO-. In addition, based on the significant advantage of dicyanoisophorone-derived fluorophore, probe NIR-PN exhibited NIR emission (lambda(em) = 660 nm) and a large Stokes shift (similar to 150 nm). Importantly, the biological application potential of probe NIR-PN was effectively verified by visualizing ONOO- levels in living cells and zebrafish.