It is intended to provide a method of reusing a DNA-immobilization substrate whereby the expensive DNA-immobilization substrate can be efficiently utilized and a reusable DNA-immobilization substrate having the same performance as a new product without any trouble in practical use can be provided. Namely, a method of reusing a DNA-immobilization substrate characterized in that, to remove DNA from a DNA-immobilization substrate carrying the DNA immobilized by an acid-amide bond via an oligonucleotide to thereby enable the immobilization of a fresh DNA, the acid-amide bond between the substrate and the DNA is hydrolyzed with an acid or an alkali.