The irregular viscosity and peroxynitrite (ONOO^–) concentration in mitochondria would result in mitochondria disorders. However, fluorescent probes that can detect both of viscosity and peroxynitrite have not been explored. For this reason, a two-photon fluorescent molecule TP-1Bz was designed and synthesized in this work for the dual-ratiometric-imaging of viscosity and exogenous and endogenous ONOO^– in mitochondria. Results indicated that TP-1Bz has two emissions at 407 nm and 650 nm respectively, their ratio (I650/I407) showed linear relationship to the viscosity of the media in the logarithmic plot, and its two-photon action cross-section (Φσ) at 880 nm was increased from 1.2 GM in aqueous solution to be 86.3 GM (72-fold) in 99% glycerol. With a good targeting capability to mitochondria, TP-1Bz allowed us to measure the average viscosity in the mitochidria of HepG2 cells (73.3 cp) and the mitochondria viscosity change upon the addition of ionpores. TP-1Bz also showed the high sensitivity to ONOO^– (LOD close to 12.1 nM) in water/glycerol (2/3) solvent as well as the high selectivity over other ROS. With the advantages of its lower cytotoxicity, pH- and photo- stabilities, TP-1Bz was successfully employed for imaging ONOO^– in living cells and even in tissue samples of rat liver through the monitoring the emission intensities of the blue and red channels. As the first example of two-photon ratiometric fluorescent probe for dual-imaging viscosity and peroxynitrite in mitochondria, TP-1Bz has great potential to be developed for clinical applications.