Soy whey (SW) is generated as a process waste during soy isolate protein preparation. SW has low content of soluble sugar and high content of oligosaccharide, which is suitable for the growth of lactic acid bacteria (LAB). In this study, a strain of Lactobacillus casei GJ00412 (by 16S rRNA genesequencing and identification) was isolated from soy whey. In order to reduce the cost of culture medium, improve its biomass, and facilitate the next utilization, Response surface methodology (RSM) was employed to optimize the composition of seed medium of Lactobacillus casei By Plackett-Burman design, sucrose, yeast power, MgSO4, Tween80 and pH value were found to have significant effects on biomass, and were further analyzed by Box-behnken design, an application of response surface. The optimized medium was as follows (w/v): 2.5% sucrose, 1.8% soy peptone, 0.4% yeast powder, 0.08% MgSO4, 0.04% MnSO4, 0.08% Tween80, pH 7.17. The results showed that the cost of the optimized medium was decreased and the biomass of Lactobacillus casei has no significant difference when compared with MRS medium.