Traditional methods of protein immobilization often result in activity loss due to random coupling. This study introduces SpyFixer, a variant of SpyCatcher that achieves over 99% efficient site-specific protein immobilization. We applied SpyFixer on two platforms: bio-layer interferometry (BLI) for protein-protein interaction analysis and epoxy agarose resin for antibody purification. Using human growth hormone (hGH) and the Z domain of Protein A as model proteins, we demonstrated that SpyFixer enables efficient, site-specific immobilization on BLI sensors, yielding reproducible kinetic data with lower variability than conventional methods. Additionally, we developed a cost-effective strategy for antibody purification utilizing SpyFixer-modified resin, which exhibited remarkable capture efficiencies exceeding 90%, elution efficiencies over 70%, and purities over 90% for human immunoglobulin G (hIgG) from complex samples, including bacterial lysates, human serum, and recombinant fermentation broth. The resin's loading capacity surpassed 200 mg/mL, and no significant activity loss was observed after 20 regeneration cycles. This study further advances the potential of Spy chemistry in biotechnological applications.