Abstract: Box-Behnken response surface method was to optimize extraction process of polysaccharide from beer vinasse and the antitumor activity of polysaccharide was evaluated.Based on the single factor experiment, leaching temperature, leaching time and solid liquid ratio as factor, the Box-Behnken central composite method was used to make three factors, three levels experimental design.The polysaccharide yield was the response value to process response surface analysis.Adopting MTT method was inspected the inhibition capability of polysaccharide from beer vinasse at 24, 48, 72 h to the cell proliferation of He La, A549 and Hep G2.It turned out that the best process of polysaccharide was when leaching temperature was 96.7 ℃, leaching time was 1.83 h, solid liquid ratio was 1∶ 27 g/m L, the maximum polysaccharide yield was 6.24%, verifying value was 6.07%, relative error was 2.72%. Polysaccharide of beer vinasse could better inhibit multiplication capacity was 34.85, 91.50 and162.39 μg/m L. It showed that the extraction process of polysaccharide from beer vinasse in the study was effective, easy and repeatable.Meanwhile, the polysaccharide could be a potential auxiliary antitumor components to apply in health food and medicine field.