The oxime-modified hierarchical self-assembly polyimide microspheres (HSPI-O) composed of polyimide nanosheets have been fabricated successfully by a solvothermal method to adsorb uranium from aqueous solution, of which the maximum adsorption capacity reaches 415 mg g⁻¹ (pH = 6). More importantly, the removal percentage of uranium in simulated uranium-containing wastewater with low uranium concentration (200–1000 μg L⁻¹) can exceed 97%, and the residual uranium concentration can satisfy the emission standard. Different from many powdery adsorbents that have been reported, the diameter of HSPI-O is relatively large (about 25 μm), and there are a large number of open spaces (about 150 nm) between the nanosheets, which are conducive to the passage of uranium-containing solution in a fixed-bed column. In addition, an eluent with a high uranium concentration is achieved with a rapid desorption process, which is conducive to the recovery of uranium. Adsorption thermodynamics and kinetic parameters reflect its adsorption is a spontaneous exothermic chemical adsorption process. The adsorption performance has almost no difference after 5 cycles, indicating a good recyclability. In summary, HSPI-O exhibits a great application potential in the fields of nuclear fuel partition and uranium-bearing wastewater remediation.