Abnormal changes occurring in the mitochondrial microenvironment are important markers indicating mitochondrial and cell dysfunction. Herein, we designed and synthesized a multifunctional fluorescent probe DPBthat responds to polarity, viscosity, and peroxynitrite (ONOO–). DPBis composed of an electron donor (diethylamine group) and electron acceptor (coumarin, pyridine cations, and phenylboronic acid esters), in which the pyridine group with a positive charge is responsible for targeting to mitochondria. D-π–Astructure with strong intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) properties give rise to respond to polarity and viscosity. The introduction of cyanogroup and phenylboronic acid esters increases the electrophilicity of the probe, which is prone to oxidation triggered by ONOO–. The integrated architecture satisfies the multiple response requirements. As the polarity increases, the fluorescence intensity of probe DPBat 470 nm is quenched by 97%. At 658 nm, the fluorescence intensity of DPBincreases with viscosity and decreases with the concentration of ONOO–. Furthermore, the probe is not only successfully used to monitor mitochondrial polarity, viscosity, and endogenous/exogenous ONOO–level fluctuations but also to distinguish cancer cells from normal cells by multiple parameters. Therefore, as-prepared probe provides a reliable tool for better understanding of the mitochondrial microenvironment and also a potential approach for the diagnosis of disease.