In order to optimize the premixing process of the herbivorous livestock manure and straw for joint photosynthetic microorganisms hydrogen production, the method of Box-Benhnken experimental design and response surface analysis were used with Design-Expert software. The response surface experiments contained three factors, such as pH value, premixed temperature and premixed time. Response value was OD value and zero point experiments proceed three times. The response surface experiment data were analyzed by Design-Expert software, and the results showed that the quadratic model was the most accurate expression. The three factors existed significant correlation. The premixing process was optimized by the amendment of the regression equation: time of 6.3d, the pH value of 4.9, temperature of 48℃, the estimated maximum OD value of 2.6968. The predicted value was closed to the average value of the validation test. The results provided a theoretical basis for the experimental study of photosynthetic microorganism hydrogen production.