The invention relates to a deer heart protein hydrolyzate, a preparation method and application thereof, and belongs to the technical field of biomedicine. The preparation method comprises the following steps: pretreatment: taking deer heart, adding physiological saline, and homogenizing tissue to obtain an enzymolysis stock solution for use; enzymolysis: taking the above enzymolysis stock solution, adding pepsin for enzymolysis reaction, and obtaining Enzymatic hydrolysis solution; purification: take the above enzymatic hydrolysis solution, separate and purify it with a Sephadex column, and take a 3KD-15KD polypeptide to obtain it. In the present invention, the heart of sika deer is used as raw material, and the small molecular polypeptide substance is obtained through tissue homogenization and a suitable enzymatic hydrolysis method. And the aging cardiomyocyte model was established, and the effect of various enzymatic hydrolyzates on the proliferation rate of cardiomyocytes was detected by MTT method. The experiment confirmed that the obtained deer heart protein hydrolyzate has good activity in alleviating the aging of cardiomyocytes.