In this study, we investigate the effect of temperature treatment on Bovine Pancreas Trypsin (BPT) in aqueous solutions using dynamic, static and electrophoretic light scattering, fluorescence spectroscopy and circular dichroism. Static and dynamic light scattering at various solution conditions i.e. different salt content and pH, reveals that BPT aggregation is enhanced as temperature increases in a non-reversible manner. At acidic pH protein monomers are the dominant population over aggregates of globules, nevertheless the two populations co-exist at neutral and basic pH. The surface charge of the aggregates is intensified by aggregation and it is dominated by the negative residues of the protein at all pH conditions. Protein unfolding upon thermal treatment is probed by variation of the fluorescence spectrum which is caused by the exposure of tryptophan to the aqueous environment. The exposure of the hydrophobic interior of BPT upon heating may be considered as the reason of aggregation at the molecular level. This study provides information that can be useful for utilizing thermal treatment protocols of BPT towards manufacturing protein-based nano formulated drugs. (C) 2019 Elsevier Inc. All rights reserved.