In mammals, a limited number of proteases catalyze with acidic amino acids as substrates. At present, there are only three known proteases: CCPs, carboxypeptidase O (CPO), and aspartate acylase (ASPA). Human CPO is a digestive enzyme that prefers glutamate as a substrate. It locates to the apical membrane of intestinal epithelial and is glycosylated protein. CPO is difficult to purify as it is a GPI-anchored protein. To obtain purified CPO, a truncated form called hCPOΔC was designed, which removed the C-terminal sequence of hCPO and was followed by His tag. Firstly, the truncated variant hCPOΔC (residues 1-349) was cloned into pFastBac vector to construct bacmid. Then the verified bacmid was transfected into Sf9 cells for expression. After the protein was successfully expressed, cell medium was collected and incubated with Ni resins. The target protein was eluted by imidazole through affinity chromatography. A purification method of human CPO with deglutamylation activity was successfully established using insect cells expression system. Purified hCPOΔC could hydrolyze glutamate in polypeptides.