Abstract: Based on bioinformatics, the amino acid residues of conservative and activity center of D-lactate dehydrogenase (D-LDH) , and the three-dimensional structure model of protein was analysised.The space conformation of visualization mutant had been constructed by homology modeling, the best mutant models were selected by the calculation of the distance and angle.The results showed that the 4 amino acid residues were relevant to the activity center in 20 conservative residues of D-LDH.After the models were compared, it was found that the big molecules substrates were obstructed by the benzyl of the residues of Phenylalanine (phe) or Tyrosine (try) on the 49 and 297 position.When F49A, Y279A, F49A and Y279A were mutated, the obstacles would disappear or weaken.The three mutants constructed were made a preliminary study, the results showed that IPTG or lactose could induce mutant to produce phenyl lactic acid in E.coli.The yield of phenyl lactic acid was higher in static culture than in vibration incubator, and the one of the F49A mutant (A.a.D-LDH-F49A strains) was higher than the one of the wild type (A.a.D-LDH strains) with lactose inducing.It would be a method of constructing gene engineering strain that visualization mutants models were compared and selected.