In living systems, proteins participate in various physiological processes and the clustering of multiple proteins is essential for efficient signaling. Therefore, understanding the effects of the number, distance and orientation of proteins is of great significance. With programmability and addressability, DNA origami technology has enabled fabrication of sophisticated nanostructures with precise arrangement and orientation control of proteins to investigate the effects of these parameters on protein-involved cellular processes. Herein, we highlight the construction and applications of protein-functionalized DNA origami nanodevices. After the introduction of the structural design principles of DNA origami and the strategies of protein-DNA conjugation, the emerging applications of protein-functionalized DNA origami nanodevices with controlled key parameters are mainly discussed, including the regulation of enzyme cascade reactions, modulation of cellular behaviours, drug delivery therapy and protein structural analysis. Finally, the current challenges and potential directions of protein-functionalized DNA origami nanodevices are also presented, advancing their applications in biomedicine, cell biology and structural biology.