Peptide molecules, due to their design flexibility, self-assembly ability, high biocompatibility, and ease of functionalization, have become versatile biomaterials for tissue engineering and biomedicine. This review highlights recent advances in the design, self-assembly, functional tailoring, and biomedical applications of peptide-based nanomaterials. The article discusses the synthesis and characterization of single, dual, and multiple stimuli-responsive peptide-based nanomaterials with various dimensions, such as pH-, temperature-, ion-, light-, enzyme-, and ROS-responsive properties. These materials are designed to respond to both internal and external stimuli, enhancing their functionality in applications like drug delivery, bioimaging, cancer therapy, gene therapy, antibacterial agents, and wound healing. The review also covers the morphological transitions of peptide nanomaterials under different stimuli, which can improve their performance in targeted therapies. Additionally, it explores the integration of active components like metal/metal oxide, DNA/RNA, polysaccharides, photosensitizers, and 2D materials to enhance the functionality of peptide nanomaterials. The comprehensive overview provides detailed methodologies and advanced techniques for the synthesis of peptide nanomaterials, aiming to guide and inspire the molecular-level design of peptides with specific and multiple functions for biomedical applications.Peptide molecules, due to their design flexibility, self-assembly ability, high biocompatibility, and ease of functionalization, have become versatile biomaterials for tissue engineering and biomedicine. This review highlights recent advances in the design, self-assembly, functional tailoring, and biomedical applications of peptide-based nanomaterials. The article discusses the synthesis and characterization of single, dual, and multiple stimuli-responsive peptide-based nanomaterials with various dimensions, such as pH-, temperature-, ion-, light-, enzyme-, and ROS-responsive properties. These materials are designed to respond to both internal and external stimuli, enhancing their functionality in applications like drug delivery, bioimaging, cancer therapy, gene therapy, antibacterial agents, and wound healing. The review also covers the morphological transitions of peptide nanomaterials under different stimuli, which can improve their performance in targeted therapies. Additionally, it explores the integration of active components like metal/metal oxide, DNA/RNA, polysaccharides, photosensitizers, and 2D materials to enhance the functionality of peptide nanomaterials. The comprehensive overview provides detailed methodologies and advanced techniques for the synthesis of peptide nanomaterials, aiming to guide and inspire the molecular-level design of peptides with specific and multiple functions for biomedical applications.