Electrostatic separation technology had been applied for the first time to the separation of algal proteins. The results indicated that grinding to a subcellular scale (<20 μm) effectively achieved the physical dispersion of Enteromorpha prolifera (E. prolifera) protein nuclei from other matrices. Compared to E. prolifera polysaccharides, proteins exhibited superior tribocharging properties, allowing them to acquire more positive charges during friction with polytetrafluoroethylene (PTFE) tubes. The electrostatic separation of E. prolifera ultrafine powder was optimized by adjusting the friction tube length and airflow velocity. It was found that with a friction tube length of 200 cm, an airflow velocity of 5 m/s, and an electric field strength of 1.2 kV/cm, the content of obtained protein increased from 4.29 ± 0.19% to 9.38 ± 0.44%, with a separation efficiency of 67.67 ± 3.14%. These findings suggest the potential application of electrostatic separation technology for the green enrichment of algal proteins.