Peptide nanozymes (PepNzymes) represent a novel class of enzyme mimics that combine the structural flexibility of peptides with the catalytic functionality of enzymes. This review explores the development, structure, functions, enzyme-like activities, and biomedical applications of PepNzymes. PepNzymes include single peptides with enzyme-like activities, peptide-based nanostructures with enzyme-like activities, and peptide-based nanozymes. These structures enable the investigation of biological phenomena at the nanoscale and offer a customizable approach that balances enzyme-like functionality with enhanced versatility, including designability, targeting, biocompatibility, and multifunctional scaffolding. The design of PepNzymes involves rational strategies such as mimicking enzyme structures, regulating peptide self-assembly, and incorporating ions, metals, or nanozymes to enhance functionality. Synthesis methods include solid-phase and solution-phase peptide synthesis, with post-synthetic modifications enabling the introduction of functional groups. Structural characterization techniques such as NMR, FTIR, CD, X-ray crystallography, and cryo-EM provide insights into the molecular structures and interactions of PepNzymes. Active groups play a crucial role in substrate recognition, binding, and catalysis, influencing the overall activity of PepNzymes. PepNzymes exhibit a wide range of enzyme-like activities, including peroxidase, oxidase, catalase, SOD, hydrolase, and isomerase activities. These activities are crucial for various biomedical applications, such as diagnostics, cellular imaging, antimicrobial therapy, tissue engineering, and anti-tumor treatments. PepNzymes also demonstrate functional diversification beyond enzyme-like activities, including biomolecular recognition, self-assembly, stimuli-responsive behavior, and biocompatibility. These properties make PepNzymes suitable for in vivo applications, such as supporting cell growth and tissue regeneration with biocompatible scaffolds or providing sustained drug release with biodegradable carriers. PepNzymes have significant potential in biomedical applications, including analysis, diagnostics, cellular imaging, antibacterial or antifungal applications, tissue engineering, and anti-tumor strategies. They enable targeted imaging and treatment of tumor cells through selective recognition and binding to cancer-specific biomarkers. PepNzymes also offer advantages in combating microbial threats by combining the antimicrobial prowess of peptides with the catalytic efficiency of nanozymes. In tissue engineering, PepNzymes can enhance the effectiveness and precision of treatments, allowing targeted delivery of therapeutic substances to specific cell populations or tissue microenvironments. Despite their promising potential, PepNzymes face challenges in structural design and fabrication, requiring optimization of nanostructure assembly details, synthetic protocols, and exploration of innovative techniques to enhance their efficacy and reliability. Addressing these challenges is essential for unlocking fresh avenues for innovative biomedical solutions and transformative progress.Peptide nanozymes (PepNzymes) represent a novel class of enzyme mimics that combine the structural flexibility of peptides with the catalytic functionality of enzymes. This review explores the development, structure, functions, enzyme-like activities, and biomedical applications of PepNzymes. PepNzymes include single peptides with enzyme-like activities, peptide-based nanostructures with enzyme-like activities, and peptide-based nanozymes. These structures enable the investigation of biological phenomena at the nanoscale and offer a customizable approach that balances enzyme-like functionality with enhanced versatility, including designability, targeting, biocompatibility, and multifunctional scaffolding. The design of PepNzymes involves rational strategies such as mimicking enzyme structures, regulating peptide self-assembly, and incorporating ions, metals, or nanozymes to enhance functionality. Synthesis methods include solid-phase and solution-phase peptide synthesis, with post-synthetic modifications enabling the introduction of functional groups. Structural characterization techniques such as NMR, FTIR, CD, X-ray crystallography, and cryo-EM provide insights into the molecular structures and interactions of PepNzymes. Active groups play a crucial role in substrate recognition, binding, and catalysis, influencing the overall activity of PepNzymes. PepNzymes exhibit a wide range of enzyme-like activities, including peroxidase, oxidase, catalase, SOD, hydrolase, and isomerase activities. These activities are crucial for various biomedical applications, such as diagnostics, cellular imaging, antimicrobial therapy, tissue engineering, and anti-tumor treatments. PepNzymes also demonstrate functional diversification beyond enzyme-like activities, including biomolecular recognition, self-assembly, stimuli-responsive behavior, and biocompatibility. These properties make PepNzymes suitable for in vivo applications, such as supporting cell growth and tissue regeneration with biocompatible scaffolds or providing sustained drug release with biodegradable carriers. PepNzymes have significant potential in biomedical applications, including analysis, diagnostics, cellular imaging, antibacterial or antifungal applications, tissue engineering, and anti-tumor strategies. They enable targeted imaging and treatment of tumor cells through selective recognition and binding to cancer-specific biomarkers. PepNzymes also offer advantages in combating microbial threats by combining the antimicrobial prowess of peptides with the catalytic efficiency of nanozymes. In tissue engineering, PepNzymes can enhance the effectiveness and precision of treatments, allowing targeted delivery of therapeutic substances to specific cell populations or tissue microenvironments. Despite their promising potential, PepNzymes face challenges in structural design and fabrication, requiring optimization of nanostructure assembly details, synthetic protocols, and exploration of innovative techniques to enhance their efficacy and reliability. Addressing these challenges is essential for unlocking fresh avenues for innovative biomedical solutions and transformative progress.