Optimization of the ultrasonic-assisted extraction of polyphenols and its antioxidant activity in Vitro from Gracilaria lemaneiformis were studied. The effects of liquid-material ratio, extract temperature, ultrasonic times on the contents of polyphenols from Gracilaria lemaneiformis were investigated. Based on this, the response surface analysis method was applied to determine the optimization of the extraction. Results showed that the optimum condition for the ultrasonic-assisted extraction of polyphenols weres as followed: ratio of liquid-material 401 (mL/g), extraction temperature 60 ℃, ultrasonic times 40 min. Under the optimum condition, the contents of polyphenols from Gracilaria lemaneiformis was 1.62 mg GAE/g. Research of its antioxidant activity in vitro shows that the polyphenols from Gracilaria lemaneiformis have scavenging capabilities of DPPH free radical and hydroxyl free radical, and their IC₅₀ are 56.67 and 18.78 μg/mL, equivalent to 15.89 and 536.4 μg/mL of ascorbic acid.