Layered nanomaterials have been recognized as promising nanomaterials for biomedical applications due to their tunable crystal phase, easy exfoliation, capability as the host to be intercalated with guest species, and layer-dependent electronic/optoelectronic properties. Recent advances in structural engineering strategies enable manipulating layered nanomaterials at the atomic level, activating and/or optimizing their properties, and overcoming existing limitations for unlocking unprecedented performance in biomedical applications. In this Review, we comprehensively summarize the latest advancements in structural engineering of layered nanomaterials, focusing on their applications in the biomedical field. First, layered nanomaterials explored in the biomedical field enabled by structural engineering are presented based on their composition and structures, followed by highlighting their unique advantages for structural engineering at the atomic level. Then, the structural engineering strategies of layered nanomaterials including crystal phase engineering, defect engineering, heteroatom doping, interlayer engineering, and crystalline-to-amorphous phase engineering are comprehensively discussed, alongside insights on the advanced characterization techniques. Moreover, the transformative potential of structural engineering to optimize the performance of layered nanomaterials for diverse biomedical applications is discussed in depth. Finally, this Review is concluded with perspectives on the key challenges and bottlenecks of structural engineering of layered nanomaterials in the biomedical field, providing potential solutions and outlining future directions.