The invention relates to the protein field, in particular to bioactive polypeptide DKLAQ as well as a preparation method and an application thereof. An amino acid sequence of the bioactive polypeptideDKLAQ is Asp-Lys-Leu-Ala-Gln. In vitro anti-oxidation experiments and in vivo anti-aging experiments prove that the polypeptide DKLAQ has the better anti-oxidation bioactivity and anti-aging activity, on one hand, the bioactive polypeptide DKLAQ has better anti-oxidation activity and can scavenge free radicals in organisms and improve the quality of life; on the other hand, the bioactive polypeptide DKLAQ can improve the vitality of in vivo anti-peroxidase systems and enhance functions of organisms for resisting exogenous stimulation to reduce the probability of organism aging, senescence anddisease and has very important significance in development of food, healthcare products and drugs with anti-oxidation and anti-aging functions.