The development of technologies for a biomedical detection platform is critical to meet the global challenges of various disease diagnoses. In this study, an inert cycloolefin polymer (COP) support was modified with two-layer polymer brushes possessing dual functions, i.e., a low fouling poly[poly(ethylene glycol) methacrylate] [p(PEGMA)] bottom layer and a poly(acrylic acid) (PAA) upper layer for antibody loading, via a surface-initiated photoiniferter-mediated polymerization strategy for fluorescence-based immunoassay. It was demonstrated through a confocal laser scanner that, for the as-prepared COP-g-PEG-b-PAA-IgG supports, nonspecific protein adsorption was suppressed, and the resistance to nonspecific protein interference on antigen recognition was significantly improved, relative to the COP-g-PAA-IgG references. This strategy for surface modification of a polymeric platform is also applicable to the fabrication of other biosensors.