The aim of this paper is to study the content and composition of hydrolyzed amino acids and free amino acids in buffalo milk and cow's milk, to provide a wider scientific basis for the development of buffalo milk products and functional foods, and to preliminarily investigate the feasibility of classifying buffalo milk and cow's milk by amino acids, which will provide a new way of thinking for the detection of adulteration in buffalo milk and cow's milk. Firstly, 30 portions of buffalo milk and 70 portions of cow's milk were collected and tested for free and hydrolyzed amino acid content using an automatic amino acid analyzer. Secondly, the amino acids of buffalo milk and cow's milk were compared in four aspects: composition, nutritional evaluation, flavor evaluation and medicinal evaluation. Finally, buffalo milk and cow's milk classification models were developed using partial least squares discriminant analysis (PLSDA) with the percentage contents of free and hydrolyzed amino acids as input variables. It was found that the total amount of free amino acids in buffalo milk and cow's milk were 120.93 μg?g-1 and 98.19 μg?g-1 milk samples, respectively, and the total amount of hydrolyzed amino acids were 4.21 g?100g-1 and 2.95 g?100g-1 milk samples; in terms of amino acid composition: the absolute content of 16 hydrolyzed amino acids in buffalo milk was significantly higher than that of cow milk (P<0.05), and the percentage content was similar to that of cow milk; except for Asp, Thr and Glu, the absolute content of the other 14 free amino acids in buffalo milk was significantly higher than that of cow milk (P<0.05), and there was a big difference in the characteristics of the percentage content; in terms of flavor and medicinal evaluation: buffalo milk In terms of flavor and medicinal evaluation: buffalo milk was rich in fresh and sweet amino acids, and the contents of essential amino acids, branched-chain amino acids and medicinal amino acids were significantly higher than that of cow's milk (P<0.05); in terms of nutritional evaluation: the EAA/TAA was 40%, and the EAA/NEAA was 76%, which was higher than the standard of ideal protein, indicating that buffalo milk and cow's milk proteins were both high-quality proteins; the nutritive value of buffalo milk proteins was higher than cow's milk in the amino acid scoring model of populations other than infants. The amino acid score of buffalo milk protein was higher than 100 under the amino acid score model of the population except infants; the accuracy of the species discrimination model was 100%. In summary, buffalo milk protein is of high value, rich in fresh and sweet amino acids that regulate the flavor of milk, as well as essential amino acids, branched-chain amino acids and medicinal amino acids that improve human body functions, and has great market potential for comprehensive development and utilization. In addition, the amino acid composition of buffalo milk and cow's milk has its own characteristics, and the utilization of amino acid characteristics can be used to identify the authenticity of the milk source.