The content of free amino acids （FAA） and hydrolytic amino acids （HAA） in 30 samples of buffalo milk and 70 samples of dairy cow milk were detected with an amino acid automatic analyzer to study the content and composition of FAA and HAA in buffalo milk and dairy cow milk. The differences in amino acid between buffalo milk and dairy cow milk were compared in terms of four aspects including the composition， nutritional evaluation， characteristics of flavor， and medicinal evaluation. A partial least squares discriminant analysis （PLSDA） was used to establish a classification model for buffalo milk and dairy cow milk with the percentage of FAA and HAA as input variables. The results showed that the total amount of FAA and HAA in buffalo milk and dairy cow milk was 120.93 μg/g and 98.19 μg/g， and 4.21 g/100 g and 2.95 g/100 g， respectively. The absolute content of 16 HAA in buffalo milk was significantly higher than that in dairy cow milk （P<0.05）， and the percentage content was similar to that in dairy cow milk. The absolute content of other 14 FAA except for Asp， Thr and Glu in buffalo milk was significantly higher than that of dairy cow milk （P<0.05）， with a big difference in the characteristics of the percentage content. Buffalo milk was rich in umami and sweet amino acids， with significantly higher content of essential amino acids （EAA）， branched chain amino acids （BCAA）， and medicinal amino acids （MAA） compared to that in dairy cow milk （P<0.05）. The ratio of EAA total amino acids （TAA） and the ratio of EAA to non-essential amino acids （NEAA） was 40% and 76%， higher than the standard of ideal protein， indicating that proteins in both buffalo milk and dairy cow milk are high-quality proteins. The nutritional value of protein in buffalo milk was higher than that in dairy cow milk. The amino acid score of protein in buffalo milk was higher than 100 under the amino acid score model of the population except infants， with the accuracy of the species discrimination model of 100%. It is indicated that buffalo milk has high value of protein， rich in umami and sweet amino acids that regulate milk flavor， EAA， BCAA， and MAA that improve functions of human body， with great market potential for comprehensive development and utilization. In addition， the composition of amino acids in buffalo milk and dairy cow milk each has its own characteristics， and the characteristics of amino acids can be used to identify the authenticity of milk sources.