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 peroxynitrite (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.