Due to their unique physical and chemical properties, two-dimensional (2D) materials are ideal carriers for single-atom catalysts. Compared with traditional one- and three-dimensional support materials, these materials not only maximize active site exposure and improve atomic utilization efficiency, but also facilitate functional modification. Their excellent cellular uptake makes them highly suitable for biomedical applications. This article summarizes recent progress in the preparation of single-atom nanozymes (SAzyme) on 2D materials. It highlights these materials' unique advantages, innovative designs, and mimicking of natural enzymes' catalytic performance. It also details the materials' specific applications and practical effects in cancer treatment, antibacterial treatment, wound healing, drug delivery, biosensing, and wastewater treatment. The potential for future clinical translation and industrial application is discussed. This review provides a theoretical basis and technical pathways for the design and application of high-performance SAzyme, thereby promoting their practical applications in the fields of biomedicine and environmental remediation.