We describe a hydrophoretic device that uses rotational flows induced by regularly patterned obstacles only on the top wall for preparing samples of biological particles, including micrometer and submicrometer particles, and DNA molecules. Many of the current continuous separation devices based on physical fields are limited to the separation of cells and micrometer-sized particles due to their dependence on a particle volume, and the purely hydrodynamic separation of macromolecules such as DNA or protein complexes remains a challenge. Hydrophoresis is entirely based on hydrodynamics using rotational flows induced by anisotropic obstacles. Different sizes of micrometer and submicrometer beads, as well as DNA molecules, were separated into distinct trajectories using two kinds of hindrance mechanisms. Continuous separation of these particles was achieved using the obstacles, demonstrating the potential of hydrophoresis for biological sample preparation on the micro- and nanoscales, with the advantages of continuous flow and sheathless passive operation.