The sequential experimental design was used to optimize the fermentation conditions of kefir from milk and soymilk.Fractional factorial design was employed to evaluate the six factors,viz.the ratio of milk and soymilk,inoculum of saccharomyces cerevisiae,inoculum of lactic acid bacteria,the addition of sucrose,fermentation temperature and time.It was found that the addition of sucrose and fermentation time were the significant factors for fermentation.Then the steepest ascent design was conducted to approach the greatest response area and the optimum combinations of significant factors were confirmed via Central Composite Design(CCD).The results indicated that the optimal conditions were the ratio of milk and soymilk was 7∶ 3,the inoculum of the LAB and yeast were 1.00×107 CFU/mL and 1.00×105 CFU/mL respectively,the addition of sucrose was 1.6%,the fermentation temperature was 22℃ and the time was 22h.