MRS culture medium was used as the basic medium for optimizing the fermented condition of Lactobacillus. rhamnosus. The four significant factors (beef extract, tween-80, peptone, diammonium citrate) were screened from these components by using a Plackeet-Burman design. Steepest ascent path was adopted to approach the optimal region, and then the Central Composite Design was used to determine the optimal levels of the main factors. Considering the fit model, cost and operation, the optimal conditions were determined as follows: beef extract 2.6%, tween-80 0.3%, peptone 2%, diammonium citrate 0.4%, with other components remaining the same, 3% inoculation, 18h culture at 37¡æ.Under such conditions, the experimental value of cell concentration were 3.54×109cfu/ml and 2.19-fold live bacteria counts in MRS-based culture medium, showing no difference with predicted value 3.68×109cfu/ml.