The objective of the present work was to investigate the physicochemical conditions that trigger the self-assembly of peptide β-lg f1-8 and therefore lead to nanofibers and hydrogel formation. Nanostructures formed by self-assembly of peptide β-lg f1-8 in the pH range of 2.0-11.0 were studied by transmission electron microscopy (TEM). Hydrogel formation was studied as a function of pH and resulted in evidence of a link between hydrogel formation and the charge distribution carried by the peptide structure. Finally, circular dichroism (CD) spectroscopy was used to characterize the effects of peptide concentration (0.4-2.0 mg/mL), ionic strength (0-1 M NaCl), and temperature (20-80 °C) on the secondary structure of peptide β-lg f1-8. Hydrogels were obtained at peptide concentrations above 2.5 mg/mL. Peptide concentration and pH adjustment were shown to trigger self-assembly of β-lg f1-8, but increasing ionic strength had no effect. Heating to 80 °C induced a stronger CD signal intensity due to an increase in solubility of the peptide, whereas only slight changes in CD pattern were found upon cooling to 20 °C. Overall, results emphasize the role of particular molecular interactions in β-sheet self-assembly of peptide β-lg f1-8 and pH-dependent electrostatic interactions occurring between β-lg f1-8 units, which can explain its propensity to self-assembly.