The invention provides a thiourea modified polyethyleneimine copolymer which is shown in a formula (I) or a formula (II). The preparation method comprises the following step: under an anhydrous condition, reacting polyethyleneimine shown in the formula (I) or the formula (II) with a compound shown in a formula (III) in an organic solvent to obtain the thiourea modified polyethyleneimine copolymer which is shown in the formula (I) or the formula (II). Due to introduction of the thiourea structure, the polyethyleneimine copolymer has low electric density, so that the cytotoxicity caused by high electric density of polyethyleneimine is reduced, and the biocompatibility is good. Meanwhile, the thiourea modified polyethyleneimine copolymer used as a gene carrier can interact with generic matters through non-charge attracted hydrogen bonds. The nanoparticles formed are slightly slack in structure. After entering cells, the nanoparticles can more effectively release the generic matters into cytoplasm and cell nucleus, so that the transfection efficiency is improved.