Abstract: Disclosed are a phosphinothricin dehydrogenase mutant, a recombinant bacterium and a one-pot multi-enzyme synchronous directed evolution method. The phosphinothricin dehydrogenase mutant, with an amino acid sequence as shown in SEQ ID No.1, is obtained by mutating alanine at position 164 to glycine, arginine at position 205 to lysine, and threonine at position 332 to alanine in a phosphinothricin dehydrogenase derived from Pseudomonas fluorescens. The recombinant bacterium is obtained by introducing a gene encoding the phosphinothricin dehydrogenase mutant into a host cell. The host cell can also incorporate a gene encoding a glucose dehydrogenase or a gene encoding a formate dehydrogenase to undergo synchronous directed evolution to achieve double gene overexpression. The one-pot multi-enzyme synchronous directed evolution method of the present invention can screen recombinant bacteria with greatly improved activity.