Milk whey proteins play a critical role in immune defense and are beneficial for human nutrition and health. The aim of this study was to characterize the milk whey proteins and their potential activities among different buffalo breeds. In this work, a tandem mass tags (TMT) proteomic approach was used to identify the differences in the proteomic profiles of milk whey proteins in Murrah, Nili-Ravi and Mediterranean water buffalos. Of the 589 identified proteins, there were 64 differentially expressed proteins identified by ANOVA analysis in buffalo milk whey proteomes. The most abundant proteins were polymeric immunoglobulin receptor, α1-antiproteinase, heat shock cognate 71 kDa protein, Acl-CoA-binding protein, pigment epithelium-derived factor, antithrombin-III, and α2-HS-glycoprotein in Mediterranean water buffalo milk, fibroblast growth factor-binding protein 1 in Murrah buffalo milk, while clusterin, actin cytoplasmic 2, peroxiredoxin-2, and sortilin in Nili-Ravi buffalo milk. Gene ontology annotation revealed that molecular function of differentially epressed proteins were protein binding, enzyme regulator activity, and molecular function regulator. Furthermore, pathway analysis indicated that most differentially expressed proteins participated in complement and coagulation cascades pathway, which are strongly related to immune function. In addition to providing insight into the complexity of the buffalo milk whey proteome and their potential physiological functions, our study provided the molecular evidence of nutritive differences among different buffalo breeds. The presence of greater abundance of immune-protection whey protein in Mediterranean water buffalo milk indicated that this high nutritive milk is a high-quality resource for dairy-based functional food exploitation.