Three-dimensional (3D) paper-based analytical devices integrate sensing and analytical functions into a unified system. This integration reflects two fundamental aspects of their 3D nature: the paper’s fibrous network structure and the dynamic operational modes of the devices. The paper’s fibrous 3D network provides capillary action, porosity, and durability, enabling fluid movement and the formation of sensing hotspots. Meanwhile, diverse 3D operational modes, such as folding, stacking, rotating, and sliding, facilitate the incorporation of multifunctional modules, simplifying complex analytical procedures for users. The combination of structural and operational features yields a cohesive and efficient 3D paper-based analytical platform. This chapter offers a detailed analysis of the design principles and characteristics of these devices, highlights key research findings, and explores their future potential.