The fractionation of bioactive peptides from hydrolysate is a main challenge to produce efficient alternative for synthetic additives. In this work, electrodialysis with ultrafiltration membrane (EDUF) was proposed to increase the purity of one antimicrobial peptide from slaughterhouse by-product hydrolysate. This targeted-peptide, α137–141 (653 Da, TSKYR), inhibits a large spectrum of microbial growths and delays meat rancidity; therefore, if concentrated, it could be used as food antimicrobial. In this context, three pH values were investigated during EDUF treatment to increase the α137–141 purity: 4.7, 6.5, and 9. pH 9 showed the highest purity increase—75-fold compared to the initial hydrolysate. Although the whole hydrolysate contains more than 100 peptides, only six peptides were recovered at a significant concentration. In this fraction, the α137–141 peptide represented more than 50% of the recovered total peptide concentration. The EDUF α137–141-enriched fraction obtained in this optimized condition would be a promising natural preservative to substitute synthetic additives used to protect food.