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Inspired by molecular self-assembly, which is ubiquitous in natural environments and biological systems, self-assembled peptides have become a research hotspot in the biomedical field due to their inherent biocompatibility and biodegradability, properties that are afforded by the amide linkages forming the peptide backbone. This review summarizes the biological advantages, principles, and design strategies of self-assembled polypeptide systems. We then focus on the latest advances in in situ self-assembly of polypeptides in medical applications, such as oncotherapy, materials science, regenerative medicine, and drug delivery, and then briefly discuss their potential challenges in clinical treatment.