SPR ligand immobilization is the core operation of immobilizing target ligand molecules on the surface of biosensor chips. Firstly, activate the carboxyl sites on the chip, bind ligand proteins or peptides via chemical bonds such as amino and sulfhydryl groups to achieve oriented or random immobilization, and then block unreacted active sites to eliminate non-specific binding. It is essential to precisely control the immobilization level and pH conditions to maintain the native spatial conformation and full biological activity of ligands, which lays a foundation for real-time binding and dissociation kinetic detection of subsequent analyte molecules. This process directly determines the stability of SPR experimental data and the accuracy of affinity determination results.