Peptide-based hydrogels have been proven to be preeminent biomedical materials due to their high water content, tunable mechanical stability, great biocompatibility and excellent injectability. The ability of peptide-based hydrogels to provide extracellular matrix-mimicking environments opens up opportunities for their biomedical applications in fields such as drug delivery, tissue engineering, and wound healing. In this review, we first describe several methods commonly used for the fabrication of robust peptide-based hydrogels, including spontaneous hydrogelation, enzyme-controlled hydrogelation and cross-linking-enhanced hydrogelation. We then introduce some representative studies on their applications in drug delivery and antitumor therapy, antimicrobial and wound healing materials, and 3D bioprinting and tissue engineering. We hope that this review facilitates the advances of hydrogels in biomedical applications.