The invention discloses a bacillus subtilis strain for producing high-purity chiral D-(-)-2,3-butanediol, and construction and applications. The construction method comprises the steps of: in bacillus subtilis, knocking out acoA gene in a degradation path of direct substrate acetoin of 2,3-butanediol, knocking out encoding gene bdhA of acetoin reductase, knocking out pta gene in a side product acetic acid formation path, knocking out ldh gene in a side product lactic acid formation path, overexpressing a synthetic branch alsSD operon by using a P43 strong promoter, overexpressing dehydrogenase encoding gene bdhA of D-(-)-2,3-butanediol by using a P43 strong promoter, and overexpressing transhydrogenase encoding gene udhA in reducing power balancing process by using a P43 strong promoter. The constructed bacillus subtilis strain is safe and harmless, and the D-(-)-2,3-butanediol, with the chiral purity of more than 99%, can be produced by utilizing glucose as a substrate.