In order to obtain high–density cultivation condition for the fermentation of Hunan brassica juncea, mixed strains of LA and R6, and single strain R2 which were suitable for the mustard starter culture, were screened out in the natural fermentation process based on indicators of sensory evaluation and nitrite content. 11 kinds of nutrients including glucose, ammonium sulfate, tryptone and beef extract were screened out as the main proliferation substances for mixed strains of LA and R6 according the result of Plackett–Burma design method; while whey powder, ammonium sulfate, tryptone and yeast extract powder were screened out for those of strain R2. The results showed that the optimum formula for mixed culture medium of LA and R6 was consisted of 3% glucose, 1.5% beef extract, 0.5% ammonium sulfate, and 1% tryptone in pH 6.0. The total number of colonies could reach to 3.62×1010 cfu/mL in the formula with conditions of 3% inoculation, 33 ℃ culture temperature, and 21 h fermentation. For strain R2, the optimum culture medium was consisted of 4% whey powder, 1.5% ammonium sulfate, 0.5% tryptone, and 2% yeast extract in pH 6.0. The total number of colonies in the formula could reach to 1.85×1012 cfu/mL at the temperature of 29 ℃ for 21 h of fermentation.