We present a three-dimensional serpentine–spiral microfluidic device that integrates inertial focusing with thermophoresis to enable efficient separation of micro- and nanoparticles. Secondary Dean flow and a controllable radial temperature gradient induce size- and thermally driven migration, extending the separation range beyond conventional single-force methods. Experiments show clear inertial focusing of microparticles and thermophoretic enrichment of nanoparticles under low Reynolds conditions. Combining both mechanisms sharpens particle focusing and enables high-efficiency sorting in mixed suspensions. Using this approach, we achieved up to 90.1% separation efficiency for 4.9 µm and 200 nm particles, demonstrating a versatile platform for biomedical and nanomaterial applications.