Donkey milk (DM) has attracted immense attention owing to its resemblance to human milk. Consequently, its components require rigorous analysis. This study aimed to identify DM serum proteins (DMSPs) using a label-free MS-based proteomics method. Herein, 1 243 DMSPs were characterized in Dezhou DM, and 788 common DMSPs were identified and subsequently analyzed. Gene ontology analysis indicated the involvement of these proteins in cellular components (extracellular exosomes, extracellular space, and focal adhesion), biological processes (small guanosine triphosphatase (GTPase)-mediated signal transduction, translation, and intracellular protein transport), and molecular functions (calcium ion binding, GTP binding, and poly(A) RNA binding). Additionally, metabolic pathway analysis confi rmed that protein processing in endoplasmic reticulum is the most related metabolic pathway, followed by those of ribosomes, phagosomes, endocytosis, complement, and coagulation cascades. This study, aimed at decoding the DM composition, advances our understanding of the potential biological functions of DMSPs.