AbstractThis paper describes a straightforward procedure to immobilize oligonucleotides on glass substrates in well‐defined micropatterns by microcontact printing with a dendrimer‐modified stamp. The oligonucleotides are efficiently immobilized by “click” chemistry induced by microcontact printing. Acetylene‐modified oligonucleotides were treated with an azide‐terminated glass slide under the confinement of the dendrimer‐modified stamp, without the use of a CuI catalyst. The immobilization is an irreversible, covalent, and one‐step reaction that results in stable attachment of the oligonucleotides. Oligonucleotides with the acetylene‐modification at the 5′ terminus hybridize selectively with full‐length, complementary targets. Strands with more than one acetylene linker do not hybridize with complementary strands.