Soups and broths are popular in the world due to their nutrition and flavor, and flavor compounds tend to be bound by the proteins in the soups and broth, influencing the flavor perception. Thus, identification of the major proteins in meat-based broth may present a basis for understanding protein adsorption of flavor compounds. The present study aimed to identify the major proteins in traditional Chinese chicken broth and to describe the structural changes of proteins during stewing (1, 2, or 3 h). As stewing time increased, protein content in the broth significantly increased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the macro-molecule proteins (>10 kDa) in the broth were mainly gelatin and actin and that the micro-molecule proteins fractions (<10 kDa) increased substantially. The gelatin had an ordered structure even after 3 h of stewing, as seen by circular dichroism (CD) spectroscopy. The presence of reactive sulfhydryl groups increased remarkably with stewing time. The surface hydrophobicity of the proteins significantly increased within 2 h then deceased slightly after 3 h. The intermolecular crosslinks, as indicated by dispersion index, increased remarkably, consistent with the result of atomic force microscopy (AFM), which together suggested that protein aggregation increased during stewing. These findings suggested that gelatin was the structural protein in the broth system and that intermolecular crosslinks functioned to maintain the broth system.