The invention discloses a chicken umami peptide and a preparation method and application thereof. The preparation method comprises the steps that chicken chest meat is taken as a raw material to be subjected to multi-enzymatic hydrolysis to obtain chicken protein enzymatic hydrolysate; the chicken protein enzymatic hydrolysate is separated and purified to obtain the novel flavor peptide, and the amino acid sequence of the peptide is pyroGlu-Cys-Gly-Gly-Ser. The chicken umami peptide has the better flavor intensity, can significantly improve the thick taste and umami of food under the concentration of 300 ppm and can be widely applied to the fields of food, seasonings and the like.