DNA origami technology enables the precise assembly of well-defined two-dimensional and three-dimensional nanostructures with DNA, an inherently biocompatible material. Given their modularity and addressability, DNA origami objects can be used as scaffolds to fabricate larger higher-order structures with other functional biomolecules and engineer these molecules with nanometer precision. Over the past decade, these higher-order functional structures have shown potential as powerful tools to study the function of various bio-objects, revealing the corresponding biological processes, from the single-molecule level to the cell level. To inspire more creative and fantastic research, herein, we highlight seminal works in four emerging areas of bioapplications of higher-order DNA origami structures: (1) assisting in single-molecule studies, including protein structural analysis, biomolecule interaction analysis, and protein functional analysis, (2) manipulating lipid membranes, (3) directing cell behaviors, and (4) delivering drugs as smart nanocarriers. Finally, current challenges and opportunities in the fabrication and application of DNA origami-based functional structures are discussed.