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Objective To optimize microwave-assisted extraction of polyphenols from Enteromorpha prolifra. Methods Based on single-factor tests, an efficient microwave-assisted extraction (MAE) technique was developed to extract bioactive polyphenols from E. prolifra through orthogonal L16(4)5 test. Results The highest yield (0.923 ± 0.013) mg/g was obtained when microwave power, solvent to raw material ratio, irradiation time, ethanol concentration, and extraction cycles were 500 W, 25 mL/g, 25 min, 40%, and 3, respectively, which was higher than that of Soxhlet extraction with methanol for 6 h, ultrasound-assisted extraction with 40% ethanol for 1 h twice and heat reflux extraction with 40% ethanol for 2 h twice. Conclusion This finding indicates that MAE is a superior technique for the extraction of polyphenols due to less impurity, higher time efficiency and yield.