Compared with conventional glass slides and two-dimensional (2D) planar microarrays, polymer-based support materials and three-dimensional (3D) surface structures have attracted increasing attention in the field of biochips because of their good processability in microfabrication and low cost in mass production, as well as their improved sensitivity and specificity for the detection of biomolecules. In the present study, UV-induced emulsion graft polymerization was carried out on a cyclic olefin copolymer (COC) surface to generate 3D nanotextures composed of loosely stacked nanoparticles with a diameter of approximately 50 nm. The introduction of a hierarchical nanostructure on a COC surface only resulted in a 5% decrease in its transparency at a wavelength of 550 nm but significantly increased the surface area, which markedly improved immobilization density and efficiency of an oligonucleotide probe compared with the functional group and polymer brush-modified substrates. The highest immobilization efficiency of the probes reached 93%, and a limit of detection of 75 pM could be obtained. The hybridization experiment demonstrated that the 3D gene chip exhibited excellent sensitivity for target DNA detection and single-nucleotide polymorphism discrimination. This one-step approach to the construction of nanotextured surfaces on the COC has promising applications in the fields of biochips and immunoassays.