Objective To establish a molecule binding model of mouse PD-1/PD-L1 protein in vitro, so as to lay a foundation for studying the biological activities of the recombinant protein and to establish a high throughput drug screening model. Methods Prokaryotic expression plasmid pET28 a (+) /mPDL-1 and pGEX-4T-1/mPD-1 were transformed to E.coli BL21 (DE3), which was then induced with IPTG. The expression products were purified by fast affinity chromatography with FPLC Protein Purification Instrument or gel separation. ELISA and GST pull-down were used to detect the interaction between mPD-1 and mPD-L1. In addition, Alamar blue was used to test the role of protein mixtures in the mixed lymphocyte proliferation. Results SDS-PAGE and Western blotting analysis showed that the recombinant proteins were successfully expressed. Then purified mPD-1 and mPD-L1 protein was obtained by affinity chromatography, Gel seperation and refolding, etc. ELISA and GST pull-down showed that mPD-1 and mPD-L1 protein had a specific binding activity in vitro. The mPD-1/mPD-L1 protein had a significantly decreased influence on the proliferation of lymphocytes（P<0.05）. Conclusion Mouse PD-1/PD-L1 recombinant protein model has been successfully established using purified mPD-1 and mPD-L1 protein expressed prokaryotically, which lays a foundation for high-throughput drug screening.