Abstract: The invention provides a recombinant bacillus subtilis, construction method and use thereof, wherein the cell's own FMMs are used as a space scaffold, and a multi-enzyme complex is constructed from specific marker proteins FloA and FloT, such that an artificial substrate channel is formed, and the cell metabolic burden is effectively reduced. The recombinant Bacillus subtilis of the invention can efficiently synthesize GlcNAc without affecting cell life activity, and can also limit the toxic intermediate metabolite GlcN-6-P near the plasma membrane to reduce or eliminate its inhibition on cell activity. In the process of shaking flask fermentation using complex medium, the yield of acetyl glucosamine of the control strain BSG-C was only 0.45 g.L?1, while that of BSG-AT, BSG-ATP, BSG-ATPB increased to 5.29 g.L?1, 6.22 g.L?1 and 8.48 g.L?1 respectively. The construction method of recombinant Bacillus subtilis is simple, easy to use and has a good application prospect.