The invention relates to the field of protein, and particularly relates to a bioactive polypeptide GLNYYQQKPVA as well as a preparation method and application thereof. The amino acid sequence of the bioactive polypeptide GLNYYQQKPVA is Gly-Leu-Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala. In vitro anti-inflammatory activity experiment and in vivo anti-aging experiment verify that polypeptide GLNYYQQKPVA has good anti-inflammatory activity and anti-aging activity, on one hand, the bioactive polypeptide GLNYYQQKPVA disclosed by the invention can promote macrophage to secrete cytokine, promote the increase of induction amount of nitric oxide of macrophage, enhance the capacity of resisting outside pathogen infection and reduce the organism incidence rate; and on the other hand, the bioactive polypeptide GLNYYQQKPVA can improve the activity of an antioxidation peroxidase system in the organism, and enhance the function of resisting exogenous stimulation of the organism, so as to reduce the possibilities of aging and disease of the organism, and thus having very important meaning for developing food, health care products and drugs with anti-oxidation and anti-aging functions.