A bacterial expression vector containing CP4-EPSPS exogenous gene was constructed and expressed efficiently in E.coli BL21（DE3）. SDS-PAGE showed that the target protein was completely soluble in the cell supernatant liquor. The supernatant was collected and further purified by nickel column affinity chromatography. The purity of CP4-EPSPS protein was above 85%, and the protein concentration was 0.9 mg/mL determined by Brandford method. The protein expression and purification system can provide stable immune antigen for the transgenic plants, food and other antibody for protein detection, and also provide a stable material basis for the preparation of protein standard material.