The invention discloses an iodine dissociation type gene cloning method based on sulfo-modifiers. According to the method, phosphodiester bonds between the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends of primers of a target gene and a plasmid vector are amplified into the sulfo-modifiers; a base sequence at the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends of forward primers of the target gene and the plasmid vector and a base sequence at the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends of reverse primers of the target gene and the plasmid vector are matched in a supplementary mode; after the target gene and the plasmid vector are subjected to PCR amplification, a PCR product is purified; iodine selectively breaks the phosphodiester bonds of the sulfo-modifiers, and under the combined action of existence of the sulfo-modifiers at the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends and iodine heat treatment, DNA will generate a 3' single-link protruding end where 20 nucleotides grow. Through the method, cloning of the target gene does not rely on restriction endonuclease, DNA ligase or any other DNA modification enzyme, operation flux is large, and the method can be applied to large-scale gene cloning.