The development of efficient adsorbents with anti-biofouling properties for uranium extraction from seawater is highly attractive to meet the growing demands for energy. However, traditional fiber adsorbents still suffer from the challenge of weak binding energy, low functional group density, and inefficient antifouling ability. Herein, inspired by the structures of phospholipid, the bio-inspired zwitterionic phosphate groups were created onto the electrospun nanofibers to manipulate the affinity with uranium and the anti-biofouling property. Additionally, hyperbranched grafting reaction together with thin diameters of the fibers were applied to significantly improve the available density of functional groups. The constructed hyperbranched zwitterionic phosphate groups functionalized nanofiber adsorbents (ZP-PAN fibers) exhibited high distribution coefficient kd (7.38 × 106 mL/g) and large adsorption capacity (1294.0 mg g−1) in the uranium-spiked seawater, surpassing most of reported polymeric adsorbents. ZP-PAN fibers also showed good selectivity against competitive cations and stable reusability. Owing to their zwitterionic structures, ZP-PAN fibers had satisfactory anti-adhesive ability toward bacteria and protein. Consequently, the uranium could be effectively extracted by ZP-PAN fibers from natural seawater with the extraction capacity of 8.1 mg g−1 after contacting with seawater for 15 days. This work demonstrates a new functionalization selection for nanofiber materials and provides a promising adsorbent in the practical uranium extraction from seawater.