We propose an innovative approach to immobilize antibodies (Abs) on solid supports, addressing challenges in generating immunosensor arrays. This method involves a self-clickable Cu2O-nanoparticle-coated silver (Cu2O@Ag) surface for boronic acid (BA) functionalization, facilitating the irreversible immobilization of native Abs. The surface catalyzes azide-alkyne cycloaddition with azido-containing BA molecules without requiring an external Cu(I) ion source. Simultaneously, a metal-enhanced fluorescence effect amplifies the array detection signal, enabling ultrasensitive serum biomarker measurement. This process improves Ab orientation, maximizing antigen-binding site exposure for target protein interaction. The Cu2O@Ag glass slides show enhanced sensitivity, reduced nonspecific adsorption, and detection limits of 0.15, 0.07, 1.70, and 0.24 ng/mL for CRP, SAP, SAA, and PSA detection, respectively, which are below the diagnostic thresholds. When validated in a multiplex format with human serum samples, the assay demonstrated strong specificity and sensitivity in detecting key cancer biomarkers (CRP, SAP, SAA, AFP, CEA, and PSA). This Cu2O@Ag strategy for nonengineered Ab immobilization has broad implications for improving affinity-based protein detection assays.