Abstract: A near-infrared region（NIR） ratiometric fluorescent probe Cy-P based on hemicyanine was rationally designed, prepared and utilized for the detection of exogenous and endogenous peroxynitrite（ONOO^-） in living cells. The research results show that Cy-P itself displays NIR emission owing to the large π-conjugated system. With the addition of the strong oxidizer ONOO^-, the π-conjugated system of Cy-P is destroyed, and a new substance with blue fluorescence is formed, leading to 268 nm blueshift of fluorescent emission. The linear detection range of the probe Cy-P to ONOO^- is 0-15 μmol/L, and the detection limit is 13 nmol/L. The probe exhibits superior selectivity for ONOO^- over various potentially interfering biological analytes. Due to the low cytotoxicity, the prepared probe is suitable for imaging detection of exogenous and endogenous ONOO^- in living cells.