The invention relates to targeted multifunctional nanoparticles and a preparation method and application thereof, and belongs to the technical field of biological medicines. The targeted multifunctional nanoparticles are prepared by an ultrasonic double-emulsification method, covalent binding is carried out by branching PEI and PLGA, PLGA nanoparticles which have negative charge originally are modified into kation nanoparticles with positive charge, plasmids can be adsorbed effectively, it ensures that the plasmid cannot drop due to physical effect in a circulation system, and after the plasmid enters cells, the possibility that the plasmid is degraded by endoenzyme is also reduced. The nanoparticles further contain IR780 which can target tumor cell mitochondria, meanwhile, by strong fluorescent light and photosensitizer characteristics of IR780, the nanoparticles become a great fluorescent and photoacoustic imaging medium, and thus, tumors can be convenient to observe. The nanoparticles can carry drug to facilitate combination with transgenic therapy, tumor cells are killed favorably, and furthermore, toxic and side effects of chemotherapy drugs can further be reduced. The preparation method of the targeted multifunctional nanoparticles is simple, is easy to operate, and is suitable for expanded production.