The technology of an immunoassay detection platform is critical to clinical disease diagnoses, especially for developing a medical diagnostic system. A polymer-based immunoassay platform was fabricated on nonwoven fabric polypropylene (PP) using a photografting reaction to graft 2-hydroxyethyl methacrylate (HEMA) and sulfobetaine (SBMA). The antifouling properties of PP-g-P(HEMA-co-SBMA) were investigated by fibrinogen adsorption and platelet adhesion. Carbonyldiimidazole was employed to activate the pendant hydroxyl groups in HEMA moieties and covalently coupled antibody molecules. The detection of the limit of the immunoassay platform was as low as 10 pg/mL. Antibody amount and bioactivity affected the availability of antibody and the sensitivity of immunoassay. The immune efficiency was dependent on the strategies of antibody immobilization. The immune efficiency of Au-g-P(SBMA-co-HEMA) and Au-SH surfaces measured by QCM-D was 165% and 35.7%, respectively. The covalently binding antibody via hydrophilic polymer chains as spacers could retain fragment antigen-binding up orientation, maintain the bioactivity of antibody, and mainly improve the accessibility of antibody molecules via adjusting the conformations of polymer chains when the antibodies recognized the antigens. Therefore, grafting hydrophilic polymers, such as zwitterionic PSBMA and reactive PHEMA onto nonwoven fabric PP, and binding antibody by covalent strategy had the potential to be developed as a commercial immunoassay platform.