Gas chromatography-mass spectrometry (GC-MS) was used to systematically compare the medium- and longchain fatty acid compositions of buffalo milk and cow milk, with the aim of identifying characteristic fatty acids in buffalo milk and establishing a method for detecting adulteration with cow milk. Differential fatty acids were screened using orthogonal partial least squares discriminant analysis (OPLS-DA), and fatty acid ratio indices were constructed based on these components. Characteristic ratio indices were subsequently selected to establish a multi-index confidence interval radar chart for the authentication of buffalo milk. A total of 50 fatty acids were detected in both milk types. The total unsaturated fatty acid content in buffalo milk (67.14%) was significantly higher than that in cow milk (61.94%) (P<0.05), whereas the total saturated fatty acid content was higher in cow milk (38.07%) than in buffalo milk (32.85%) (P<0.05). Based on the OPLS-DA model, 23 differential fatty acids (VIP>1) were identified, among which 13 fatty acids, including C22:1T, C20:3, and C22:5n-6, were significantly more abundant in buffalo milk than in cow milk (P<0.05). The confidence intervals of the two fatty acid ratio indices, C13:0/C22:5n-6 and C14:1/C22:5n-6, did not overlap and exhibited the largest differences between the two milk types. These results indicate that the two ratio indices can effectively distinguish buffalo milk from cow milk under the experimental conditions applied. The identified fatty acids and ratio indices show potential as reference markers for the detection of buffalo milk adulteration and may provide practical value for milk quality authentication.