Peptides can form nanofibers of a certain conformation through noncovalent interactions, and these nanofibers are entangled and cross-linked together to form peptide hydrogels with a three-dimensional (3D) porous structure. The controllability and biocompatibility of the peptide self-assembly (PSA) endow peptide hydrogels with a wide range of applications in the fields of biomedicine and tissue engineering. Therefore, the in-depth understanding of the influencing conditions and properties of the PSA to form hydrogels is of great significance for the design and preparation of biomedical nanomaterials. In this chapter, we focus on the factors that induce the PSA to form 3D hydrogels and give a brief overview of the rheological properties and biocompatibility of peptide hydrogels, and look forward to the future development of peptide hydrogels in the field of nanobiotechnology and materials science.