The invention discloses a method for improving yield of butanol produced by escherichia coli. According to the method for constructing recombinant bacteria, expression of a malate dehydrogenase gene (mdh) on the genome of an original strain for producing butanol is inhibited or silenced to obtain the recombinant bacteria. Experiments prove that the modified target capable of promoting escherichia coli to produce butanol is the malate dehydrogenase gene (Genbank: mdh gene (b3236) in the NC_000913.3 sequence). By a lembda-red homogenous recombinant system knockout technology, after the mdh gene of the engineering strain of escherichia coli for producing butanol is knocked out, the butanol output can be increased by 283 percent, and the yield can be increased by 89 percent.