The invention provides a cutlassfish antioxidative peptide isolation and purification method, comprising the steps of 1) pretreating; 2) enzymatically hydrolyzing; 3) ultrafiltering; 4) performing QFF (Q sepharose fast flow) anion exchange chromatography; 5) performing gel chromatography; 6) performing high-performance liquid chromatography. The method has the advantages that in QFF anion exchange chromatography, Tris-HCl buffer solution and sodium borate are added in QFF elution, column hanging rate of proteins is effectively increased, it is avoided that part of the proteins are eluted off while NaCl is not added, waste of active proteins is avoided, and extraction rate of the active proteins is increased; eluting speed is high, energy is saved, manpower and time costs are decreased, and the quality of active peptides is maintained effectively; finally obtained active peptides gain greatly improved DPPH (1,1-diphenyl-2-picryl-hydyazyl) free radical removing ability, hydroxy free radial removing ability, reducing capacity and superoxide anion removing ability as compared with initial hydrolysates.