Abstract: With Grateloupia livida as the raw material and protein extraction rate as the index, we explored the effect of different wall-breaking techniques on their protein extraction, and optimized the ultrasonic-assisted water extraction by single-factor analysis and orthogonal test. The antioxidant activity of the crude protein that was purified by ammonium sulfate salting-out was evaluated. Results show that the highest protein extraction rate of the swelling method, freeze-thawing method and bead milling method was (29.66±0.86) %, (24.52±0.04) % and (26.52±0.79) %, respectively, all lower than those of ultrasonic-assisted water extraction method. The best optimal conditions of extracting protein from Grateloupia livida by ultrasonic assisted water extraction (the protein extraction rate could reach 58.16%) are as follows: liquid-to-solid ratio of 160 mL·g⁻¹, total ultrasonic time of 60 min, ultrasonic power of 1440 W and pH of 6.0. The crude protein had strong antioxidant activity at the concentration of 1−8 mg·mL⁻¹ (its reducing power at 8 mg·mL⁻¹ was 0.43±0.01), and the IC₅₀ of DPPH free radicals and ABTS free radicals are 4.00 mg·mL⁻¹ and 3.96 mg·mL⁻¹, respectively.