The invention relates to the field of proteins, and particularly relates to a bioactive polypeptide EVIESPPEINTV, and a preparation method and application thereof. The amino acid sequence of the bioactive polypeptide EVIESPPEINTV is Glu-Val-Ile-Glu-Ser-Pro-Pro-Glu-Ile-Asn-Thr-Val. Through an in-vitro immune function regulation experiment and an in-vivo anti-aging experiment, the polypeptide EVIESPPEINTV is verified to have a relatively good immune adjusting function and anti-aging activity. On one hand, the bioactive polypeptide EVIESPPEINTV provided by the invention can enhance the in-vitro proliferation capability of lymphocytes and macrophages, the ability for resisting external pathogen infection of an organism is improved, and the morbidity of the organism is reduced; on the other hand, the activity of an anti-peroxidation enzyme system in vivo can be improved, the function of resisting exogenous stimulation of the organism is enhanced, so that the probability of aging, senility and disease of the organism is reduced. The bioactive polypeptide EVIESPPEINTV has important significance for developing foods, health products and medicines with an immunomodulatory function and an anti-aging function.