Abstract2D materials have offered essential contributions to boosting biocatalytic efficiency in diverse biomedical applications due to the intrinsic enzyme‐mimetic activity and massive specific surface area for loading metal catalytic centers. Since the difficulty of high‐quality synthesis, the varied structure, and the tough choice of efficient surface loading sites with catalytic properties, the artificial building of 2D nanobiocatalysts still faces great challenges. Here, in this review, a timely and comprehensive summarization of the latest progress and future trends in the design and biotherapeutic applications of 2D nanobiocatalysts is provided, which is essential for their development. First, an overview of the synthesis‐structure‐fundamentals and structure‐property relationships of 2D nanobiocatalysts, both metal‐free and metal‐based is provided. After that, the effective design of the active sites of nanobiocatalysts is discussed. Then, the progress of their applied research in recent years, including biomedical analysis, biomedical therapeutics, pharmacokinetics, and toxicology is systematically highlighted. Finally, future research directions of 2D nanobiocatalysts are prospected. Overall, this review to provide cutting‐edge and multidisciplinary guidance for accelerating future developments and biomedical applications of 2D nanobiocatalysts is expected.