rCCK8PE38 is a novel immunotoxin that targets choleystokinin B receptor (CCKBR), which is over-expressed in some tumour tissues. Although we constructeda prokaryotic expression vector to express rCCK8PE38 in our lab, thorough purification was necessary to quantitatively assess its anti-tumour effect. In this study, we established a purification protocol to obtain rCCK8PE38 with high purity from E. coli. Three different types of chromatography, including hydrophobic chromatography (HIC), ion exchange chromatography (IEC) and size exclusion chromatography (SEC), were used in combination. The purification technological parameters of each chromatography type were optimized. The whole process of purification was arranged to minimize the purification steps and achieve purity andbioactivity. Finally, through this optimized scheme, we obtained a recombinant protein with a purity of >95 %; then,the protein was stored at -80 °C after lyophilisation. The purified protein was used in a tumour inhibition experiment and was effective in killing tumour cells that over-expressed CCKBR. The results of this study may provide some valuable information about protein purification and lay the foundation for further clinical experiments with rCCK8PE38.