Objective： To evaluate the removal effect of endotoxin in purification process of Cu， Zn-SOD expressed in recombinant Pichia pastoris. Methods： The active carbon adsorption and Triton X-114 phase separation were separately or jointly adopted to remove the endotoxin from Cu， Zn-SOD protein solution expressed in recombinant Pichia pastoris. Results： Both methods worked well but with different efficiency. Removal rate of endotoxin was 99.8% with the protein recovery rate of 66% after active carbon absorption combined with Trition X-114 phase separations. The endotoxin content of the final product was reduced to 26.8 EU/10 000 U determined by Limulus amebocyte lysate （LAL） assay. The residue of Triton X-114 in protein solution was 5.7 μg/mL measured by HPLC chromatography method. Conclusions： Active carbon adsorption combined with Triton X-114 phase separation is a better and economic method to purify the recombinant proteins and remove endotoxin in large scale. Both endotoxin and Triton X-114 in the purified recombinant Cu， Zn-SOD protein solution were effectively removed and their residues were well below the required limits of Chinese Pharmacopoeia.