The invention relates to an ABC-type amphiphilic biodegradable polyester triblock copolymer and a preparation method and application thereof. In the ABC-type polyester triblock copolymer, an A block is polyethylene glycol monomer units, a B block is hydrophobic degradable polyester, is lactone, lactide or copolymers thereof, and a C block is homopolymers or copolymers of monomers capable of performing atom transfer radical polymerization; and the ABC-type amphiphilic polyester triblock copolymer can be self-assembled to form spherical nanoparticles in water, is used for hydrophobic medical nano/micron carriers, serves as DNA, RNA carriers and is used for pharmaceutical preparations, cell transfection reagents, detection reagents and immune preparations. The polymer is biodegradable in vivo, the degradation products are non-toxic and harmless and have water absorption, permeability and biocompatibility, and the invention is easy and convenient to operate and can be applied to medicament delivery, tissue engineering and other biomedical fields.