The objectives of the present study were to examine the antioxidant activity of fractions with different isoelectric points from salmon enzymatic hydrolysates and obtain peptide fractions of sufficient amounts with higher antioxidant peptide fraction, which could be applied to the food and animal model systems. The salmon enzymatic hydrolysates were fractionated on the basis of the amphoteric nature of sample peptides by preparative isoelectric focusing without toxic solvents and reagents, which is termed autofocusing. Acidic and basic fractions showed higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than the other fractions. The basic fractions showed higher hydroxyl (OH) radical scavenging activity. The weak acidic and weak basic fractions showed higher oxygen radical absorbance capacity (ORAC) values than the acidic and basic fractions. The acidic fraction showed higher metal chelating activity than other fractions. The acidic fraction suppressed lipid oxidation in the cooked patties to a greater extent than other fractions. These results suggest that peptides fractions from salmon enzymatic hydrolysate are effective antioxidant, and that autofocusing could be useful to increase antioxidant activity for application to food and animal model systems.