In order to realize the mechanism of aggregate formation of proteins in solution, the thermodynamic properties and state diagram of the thermally induced aggregation of protein at various molar fractions of 1,4-dioxane were determined by DSC, circular dichroism (CD), dynamic light scattering (DLS), and scanning electron microscopy (SEM). In this solution, the transition temperatures of thermal denaturation and aggregation differ significantly, so each transition enthalpy can be easily determined. The transition temperature and enthalpy of protein were significantly dependent on the molar fraction of 1,4-dioxane, incubation time, and scan rate. In this paper, the DSC measurements for protein aggregation are introduced focusing on the recently revealed results for β-lactoglobulin (LG). The secondary structure of LG influenced greatly with dioxane concentration, temperature, and incubation time, resulting the aggregated structure became spherical or amorphous. The enthalpy changes for the thermal aggregation were large negative (exothermic) which may reflect to the aggregation from a supersaturated state of the protein and the interaction between protein molecules.