β-lactoglobulin (BLG), the major bovine whey protein, is a well-characterized globular protein. It is a model protein for studying the structural transition and aggregation. BLG unfolds and aggregates through chemical and physical processes. It is a predominantly β-sheet protein but, the non-native α-helical intermediate accumulates in its folding pathway. The present study aims to understand more about which stage of the protein folding is prone to aggregation. The intermediate states were trapped by TFE and their aggregation and structural changes evaluated, for this purpose. The experiments were carried out at various pH values, ionic strengths, protein concentrations and heating times by turbidity measurements, circular dichroism and fluorescence spectroscopy. Furthermore, the aggregated species at various molecular weights were detected by SDS-PAGE. Only a small change was observed in the secondary and tertiary structures of the protein at 10% TFE, but a further increase of TFE concentration results in induction of new α-helical structure and disruption of the rigid tertiary structure. The turbidity measurement indicated that the aggregation of BLG reaches a maximum level at 10% TFE on all experimental conditions and from this point forward, it decreases with increasing the amount of TFE. In conclusion, the results showed that the α-helical state is resistant to aggregation, in spite that its tertiary structure is partially unfolded. BLG becomes prone to aggregation, when its non-native α-helical structure converts to the β-sheet structure.