Peptide-based hydrogels form a kind of promising material broadly used in biomedicine and biotechnology. However, the correlation between their hydrogen bonding dynamics and mechanical properties remains uncertain. In this study, we found that the adoption of β-sheet and α-helix secondary structures by ECF-5 and GFF-5 peptides, respectively, could further form fiber networks to immobilize water molecules into hydrogels. Increasing the peptide concentrations improvethe solidity of these hydrogels, as evidenced by their higher storage modulus (G') values determined by a frequency sweep. Raman and FTIR spectroscopies probed a blue shift in the O-D stretching vibration in both ECF-5 and GFF-5 hydrogels, indicating the D-O bond contraction and stiffness gain. This finding provides valuable insight and offers an efficient means of modulating the mechanism of mechanical properties.