The invention relates to a method for preparing dTDP-L-rhamnose in a one-pot-four-enzyme mode.The method comprises the following steps that 1, a reaction system with deoxyribose thymidine triphosphoric acid, Glc-1-P, MgC12, pyrophosphoric acid hydrolytic enzyme, NAD+, NADH and synthetase Cps23FL; 2, the reaction system is subjected to a reaction at the temperature of 16-38 DEG C, then synthetase is added, the reaction continues to be carried out, and a reactant solution is obtained; 3, the reaction solution is ultrafiltered, purified and dried to prepare the dTDP-L-rhamnose.The dTDP-L-rhamnose synthetase from the streptococcus pneumoniae 23F type is utilized for synthesize dTDP-L-rhamnose in vitro through an enzymatic method.The purified synthetase is high in purity, has the exclusive substrate recognition ability and catalytic reaction efficiency and is easy to prepared, and therefore the problem that crude enzyme liquid has complex ingredients in a traditional enzymatic synthesis method is avoided.