The invention discloses a mitochondrion 3,4,9,10-perylenetetracarboxylic dianhydride targeting fluorescent probe and an application thereof. The molecular formula of the mitochondrion 3,4,9,10-perylenetetracarboxylic dianhydride targeting fluorescent probe is C104H108Br2N4O18P2, and the molar mass of the fluorescent probe is 1923.74. The mitochondrion 3,4,9,10-perylenetetracarboxylic dianhydride targeting fluorescent probe is characterized in that two sides of a fluorophore 3,4,9,10-perylenetetracarboxylic diimide are connected with two water-soluble PEO chains, and the hydrophilicity of the probe is improved greatly; by means of presence of a linking group, the whole conjugate plane is enlarged, and the fluorescence property of the probe is improved; a triphenylphosphine cation is introduced to the position of the linking group, so that the linking group serving as a targeting group can be specifically bound to the mitochondrion. The invention further provides the application of the mitochondrion 3,4,9,10-perylenetetracarboxylic dianhydride targeting fluorescent probe in living cell imaging and provides an application method of the mitochondrion 3,4,9,10-perylenetetracarboxylic dianhydride targeting fluorescent probe. Experiments prove that the fluorescent probe enters cells easily, is good in cell uploading rate and good in cell permeability and cannot produce cytotoxicity.