The YAFAF-based hydrogel was a three-dimensional network cross-linked by grooved fiber bundles. The fiber bundles were formed by entanglement of fibrils with a diameter of 2 nm, and the surface of the fibrils also presented grooves. Spectroscopic analysis revealed that the main secondary structures were β-sheets and β-turns, which led to the grooved feature of fibrils. In comparison of the nuclear magnetic resonance spectra of peptide solutions at 313 and 277 K, the nuclear Overhauser effects can be clearly observed, indicating that hydrogen-bondings and π-π stacking interactions play important roles in self-assembly. The micro-organization of the self-assemblies was affected by the ratio of solvents ( x A ) remarkably. Unexpectedly, x A of 0.05 produced hollow spherical aggregates. The result of these investigations on the mechanism and organization of the YAFAF-based hydrogel can contribute to the development of strategies using hydrogels in the food industry.