Abstract：Bacillus amyloliquefaciens CQN-2 was an endogenous bacteria isolated from the intestinal tract of Apostichopus japonicus, which showed many biological activities such as disease resistance and growth promotion. To investigate the optimal culture medium components and fermentation conditions, the bacterial biomass was used as indicator to determine the optimal conditions of CQN-2 strain growth by single factor test and orthogonal experiment design. The optimal nutritional conditions and optimal fermentation conditions were screened. The results showed that the optimum carbon source of CQN-2 strain was soluble starch, and the optimum nitrogen source was bacteriological peptone. The optimal medium composition was as followed: soluble starch 20 g/L, bacteriological peptone 30 g/L, molasses 50 g/L, KH2PO4 0.5 g/L, MgSO4 0.5 g/L, NaCl 0.3 g/L, Mn2+ 5 mmol/L and Al3+ 1 mmol/L. The optimum fermentation conditions were initial pH 7.0, inoculation rate was 3%, and medium capacities was 25 mL/250 mL. Then the strain CQN-2 was prepared with high density fermentation in 5 L fermentation tank under optimized conditions for 36 h, while the viable cell count of strain CQN-2 was 3.03×1012 cfu/mL.