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.　©　2019,　Editorial　Office　of　Journal　of　CIFST.　All　right　reserved.