A preparation method of labeled DNA and use thereof are disclosed. More specifically, the invention relates to a preparation method of labeled DNA by preparation of a complex triple-stranded DNA through an deoxyoligonucleotide complementary to the deoxyoligonucleotide sequence of the 3′ terminal sequence of a certain double-stranded DNA followed by replacement of the 3′ terminal region of the plus strand of said double-stranded DNA with at least one labeled dNTP, and also to said immobilization method of a DNA obtained from the preparation method of the labeled (or modified) DNAs onto a solid support. These subject matters are mainly useful for preparation of labeled DNA molecules.