Biomolecular motors are dynamic systems found in organisms with high energy conversion efficiency. FOF1-ATPase is a rotary biomolecular motor known for its near 100% energy conversion efficiency. It utilizes the synthesis and hydrolysis of ATP to induce conformational changes in motor proteins, thereby converting chemical energy into mechanical motion. Given their high efficiency, autonomous propulsion capability, and modifiable structures, FOF1-ATPase motors have attracted significant attention for potential biomedical applications. This Review aims to introduce the detailed structure of FOF1-ATPase, explore various motility manipulation strategies, and summarize its applications in biological detection and cargo delivery. Additionally, innovative research methods are proposed to analyze the motion mechanism of FOF1-ATPase more comprehensively, with the goal of advancing its biomedical applications. Finally, this Review concludes with key insights and future perspectives.