Nuclear energy plays a key role in the global energy supply. The adsorption and extraction of uranium from seawater is key to the rapid development of nuclear energy. In this paper, quaternary aminated polyethyleneimine/amidoxime-modified hemp fibers (HFAO-QPEI) with high adsorption capacity and anti-biofouling properties were successfully synthesized by immersion and hydrothermal methods. Upon systematic evaluation, HFAO-QPEI possessed high adsorption capacity and good reusability with fast adsorption kinetics, as well as good adsorption selectivity and affinity for uranyl ions. Furthermore, HFAO-QPEI not only reached a maximum adsorption capacity of 364.7 mg/g but also demonstrated a short adsorption equilibrium time of 200 min. In contrast, HFAO-QPEI demonstrated high selectivity for uranyl ions, removing up to 95.1 % of uranium with Kd value of 4.69 × 104 mL/g, which was 256.3 times greater than that of hemp fibers (HF). After the fifth adsorption–desorption cycle, the recovery of U(VI) was 91.38 %. DFT calculations proved that HFAO-QPEI-U was the most stable model. The quaternary ammonium salt functionalization not only makes HFAO-QPEI have excellent anti-biofouling ability (anti-diatom rate up to 87.22 %, anti-bacterial rate up to 97 % or more, anti-adhesion of large fouling organisms mussels with a certain effect), but also modifies its surface charge, making it more suitable for the marine environment. In addition, HFAO-QPEI showed a high adsorption capacity of 2.45 mg/g in natural seawater. The results suggest that the HFAO-QPEI adsorbent holds great promise for extracting uranium from seawater.