Fabrication of three-dimensional (3D) surface structures for the high density immobilization of biomolecules is an effective way to prepare highly sensitive biochips. In this work, a strategy to attach polymeric microspheres on a cyclic olefin copolymer (COC) substrate for the preparation of a 3D protein chip was developed. The COC surface was firstly functionalized by the photograft technique with epoxy groups, which were subsequently converted to amine groups. Then monodisperse poly(styrene-alt-maleic anhydride) (PSM) copolymer microspheres were prepared by self-stabilized precipitation polymerization and deposited as a single layer on a modified COC surface to form a 3D surface texture. The surface roughness of the COC support undergoes a significant increase from 1.4 nm to 37.1 nm after deposition of PSM microspheres with a size of 460 nm, and the modified COC still maintains a transmittance of more than 63% at the fluorescence excitation wavelengths (555 nm and 647 nm). The immobilization efficiency of immunoglobulin G (IgG) on the 3D surface reached 75.6% and the immobilization density was calculated to be 0.255 μg cm-2, at a probe protein concentration of 200 μg mL-1. The 3D protein microarray can be rapidly blocked by gaseous ethylenediamine within 10 minutes due to the high reactivity of anhydride groups in PSM microspheres. Immunoassay results show that the 3D protein microarray achieved specific identification of the target protein with a linear detection range from 6.25 ng mL-1 to 250 ng mL-1 (R2 > 0.99) and a limit of detection of 8.87 ng mL-1. This strategy offers a novel way to develop high performance polymer-based 3D protein chips.