The invention relates to the technology in the field of food and beverage processing, in particular to preparation of an egg and milk essence substrate by using a dual enzymatic method in cooperation with the mailard reaction; and the prepared egg and milk essence substrate can be widely applied to various food industries. The method comprises the following steps of: hydrolyzing natural cream by using lipase and subjecting dried egg yolk to enzymolysis by using protease; mixing a hydrolysis product and an enzymolysis product based on a certain ratio, homogenizing by a homogenizer, and obtain a homogenous emulsification system by using the monoglyceride and lecithin in a mixture per se as emulsifiers; then adding reducing sugar-xylose and amino acid-methionine for producing the egg characteristic flavor, mixing to reach a uniform state, directly heating to 105-110 DEG C, reacting for 1-3 hours; and performing the mailard reaction and enzyme deactivation at the same time. A final product has the flavor mixture of free fatty acid, lactone, methyl ketone, alcohol, aldehyde, black-like essence with antioxidant activity and the like, wherein the free fatty acid is obtained through hydrolyzing the fat, the lactone, methyl ketone, alcohol and aldehyde are obtained through heating the fat, and the black-like essence is generated by the mailard reaction; and thus, the product has an intense egg and milk flavor.