Abstract: Extraction of polyphenols from Humulus lupulus L was optimized from extracting solvents and other extracting conditions. The results indicated that the mixture of acetone and water was the best solvent. The results of fractional factorial design experiment showed that the factors such as acetone concentration, extracting temperature, material and solvent ratio, extracting time had effects on the extracting rate. Acetone concentration and extracting temperature were the most significant factors for extracting rate compared with the other factors. The optimal condition described as follows: acetone concentration was 62% and extracting temperature was 47℃ by the steepest ascent experiment and the central composite design. Under the optimized condition, the highest extracting amount of polyphenols from Humulus lupulus was 47.40 mg·g^-1.