The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating the efficacy of PD-1/PD-L1 inhibitors. A previously established human PD-1/PD-L1 blockade assay utilizing Surface Plasmon Resonance (SPR) technology (first-generation PD-1/PD-L1 inhibitor SPR screening platform) demonstrated results comparable to those obtained through Homogeneous Time-Resolved Fluorescence (HTRF) and cell-based assays, with potential for large-scale screening. Herein, an optimized version of this assay (second-generation PD-1/PD-L1 inhibitor SPR screening platform) is presented, featuring a dual-step coupling process that combines amine and bio-streptavidin coupling to enhance PD-1 orientation control on the chip and reduce PD-1 protein consumption. The updated platform was successfully validated using the PD-1/PD-L1 inhibitor BMS-1166, showing blockade effects comparable to the previous SPR-based method and other established techniques such as ELISA. These results confirm the reliability of the approach. This optimized SPR screening platform offers a high-throughput and reliable tool for identifying novel PD-1/PD-L1 inhibitors, advancing cancer immunotherapy research, and highlighting the potential of SPR in immune checkpoint inhibitor screening.