Key enzyme genes: glycerol dehydrogenase (gdh) and acetoin reductase (acr), which participate in coenzyme cyclic regeneration in 2,3-butylene glycol synthesis, and a regulating factor (aslR) for regulating enzyme activity of key enzyme in branch route of 2,3-butylene glycol synthesis are cloned from Bacillus subtilis CTCC M2009200; then the genes are connected to a shuttle expression plasmid (pMA5-HpaII) to successfully construct a shuttle expression plasmid with gdh, acr, and aslR (pMA5-HpaII-acr-HapII-gdh-PbdhA-alsR), and then pMA5-HpaII-acr-HapII-gdh-PbdhA-alsR is transferred into Bacillus subtilis CTCC M2009200 to obtain recombinant bacillus subtilis with enhanced gene expression of gdh, acr, and alsR, wherein the recombinant bacillus subtilis is named as GAR. The original bacterium and recombinant bacterium (GAR) are used to ferment glycerin to produce 2,3-butylene glycol; the results show that compared with original bacterium, the output of 2,3-butylene glycol is increased by 17.2%, in the recombinant bacterium (GAR) group, moreover, the outputs of byproducts: acetoin, lactic acid, and succinic acid are reduced by 69.2%, 45.2%, and 47.4% respectively, and the fermentation period is shortened. By supplementing raw materials and adding glycerin, after 48 hours of fermentation, the accumulated amount of 2,3-butylene glycol in fermentation broth can reach 102.6 g/L, and the accumulated amount cannot be achieved by a conventional technology.