The change in the level of ONOO− is usually indicative of an abnormality in bodily function. Therefore, there is a need to develop highly reliable ONOO− assays to determine its role in the biological environment. Thus, a long- wavelength anthracycline-based fluorescent probe (LAP) with a new reaction site was presented to detect per- oxynitrite (ONOO− ) by one step synthesize between 6-hydroxy-1-tetralone and salicylaldehyde. LAP responded to ONOO− about 8-fold fluorescence changes at 628 nm and the detection limit was 3.7 nM. The ONOO− sensing of LAP with the disruption of the conjugated skeleton was highly specific and could identify ONOO− by naked eyes. LAP was specifically localized to the mitochondria and co-localization results of LAP and MitoTrancker Green showed that the merged fluorescent images were observed with high Pearson’s co-localization coefficient 0.92 and 0.90 in SMMC-7721 and RAW264.7 cells, respectively. As a specific and sensitive fluorescence probe, LAP with mitochondria-targetable moiety was ability to detect exogenous ONOO− in SMMC-7721 and RAW264.7 cells.