To improve the collagen production of recombinant Pichia pastoris during fermentation of induction phase, in this paper, the Placket-Burman and Box-Behnken design were applied to optimize the fermentation medium (BMMY). Three variables (the concentration of yeast extra, methanol and ammonia sulfate) which were proved to have significant effects on the production of collagen were selected from six variables by Placket-Burman design. Through Box-Behnken design (BBD) regression coefficients analysis, a secondary degree polynomial equation was established, and the optimum levels of the three variables were as following: yeast extra 1.19%, peptone 1.18% and glycerol 0.77%. From the 3D response surface plots and 2D contour plots created by the BBD, it is significantly observed the interaction between yeast extra and methanol or yeast extra and ammonia sulfate. An average production of 5.02 g/L after 84 h cultivation in the optimized BMMY media could be attained by the validation experiments, which was 19.3% higher than the formal yield of 4.05 g/L.