Abstract: Synthesis of N-acetylneuraminic acid using Bacillus subtilis is favorable due to high expression level and the fact that B.subtilis meets the strict safety criteria for application in food industry. In this study, a recombinant Bacillus subtilis was constructed and used for fermentation studie in N-acetylneuraminic acid production. First, a unique promoter P_holinoriginated from B.subtilis lysogen was optimized for heterologous transcription, which level was evaluated by comparison study together with other promoters routinely used in Bacillus. The results showed that GFP fluorescence level from recombinant strain containing plasmid p MK4-P_holin-GFP was 2.62 times of that obtained by promoter P43.The acetylneuraminic acid expression cassette neu BC was then inserted behind P_holinin p MK4 to construct p MK4-P_holin-neu BC, which was transformed into B.subtilis 168.The recombinant B. subtilis was able to produce up to 0.226 g/L N-acetylneuraminic acid in fermentation study using shaking flasks, which laid a solid basis for further strain optimization and industrial production of N-acetylneuraminic acid.