High-throughput, high-precision single-stream focusing of microparticles has a potentially wide range of applications in biochemical analysis and clinical diagnosis. In this work, we develop a sheathless three-dimensional (3D) particle-focusing method in a single-layer microchannel. This novel microchannel consists of periodic high-aspect-ratio curved channels and straight channels. The proposed method takes advantage of both the curved channels, which induce Dean flow to promote particle migration, and straight channels, which suppress the remaining stirring effects of Dean flow to stabilize the achieved particle focusing. The 3D particle focusing is demonstrated experimentally, and the mechanism is analyzed theoretically. The effects of flow rate, particle size, and cycle number on the focusing performance were also investigated. The experimental results demonstrate that polystyrene particles with diameters of 5–20 μm can be focused into a 3D single file within seven channel cycles, with the focusing accuracy up to 98.5% and focusing rate up to 98.97%. The focusing throughput could reach up to ∼105 counts/min. Furthermore, its applicability to biological cells is also demonstrated by 3D focusing of HeLa and melanoma cells and bovine blood cells in the proposed microchannel. The proposed sheathless passive focusing scheme, featuring a simple channel structure, small footprint (9 mm × 1.2 mm), compact layout, and uncomplicated fabrication procedure, holds great promise as an efficient 3D focusing unit for the development of next-generation on-chip flow cytometry.