An object of the present invention is to develop and provide a method for efficiently and conveniently producing a nucleic acid aptamer, particularly, a DNA aptamer, having high specificity for and high binding activity against a target substance. The present invention provides a method for producing a nucleic acid aptamer, comprising: a complex formation step of mixing a single-stranded nucleic acid library with a target substance in a solution to form a complex of a single-stranded nucleic acid and the target substance； an immobilization step of mixing the solution after the preceding step with a solid-phase support to immobilize the complex onto the solid-phase support via connector(s) adsorbed on the target substance and/or the solid-phase support； a recovery step of recovering the complex immobilized on the solid-phase support from the solution； an amplification step of recovering the single-stranded nucleic acid from the complex, followed by amplification by a nucleic acid amplification method； and a single-stranded nucleic acid preparation step of converting the double-stranded nucleic acids obtained in the amplification step into single strands and then forming an intramolecular conformation.
