Pyridoxal 5’-phosphate (PLP) is a cofactor utilized by over 300 enzymes for the conversion of enormous valuable products in pharmaceutical, food, and chemical industries. Many PLP-dependent enzymes are homodimeric with catalytic pockets concaved by two subunits. To tailor the activity of these PLP-dependent enzymes, previous studies usually targeted the substrate-binding pocket or transition states. In this work, we selected two different PLP-dependent enzymes, PjAT ( Pseudomonas jessenii ω-aminotransferase) and ODC (ornithine decarboxylase), to study how subunit-subunit affinity affects their enzyme activity. We found that mutants of PjAT and ODC with low subunit-subunit affinities could have high activity. Among protein homodimerization, substrate binding, and catalytic turnover, the last one was rate-limiting. Furthermore, the activity change of these enzymes from interface engineering was substrate independent. Combining the interface engineering strategy with the design of catalytic pockets and transition states further improved the activity of these PLP-dependent enzymes. Therefore, for the first time to our best knowledge, our work revealed that subunit-subunit interface engineering is a worthwhile tool to use in the design PLP-dependent enzymes. Our work expanded the toolbox of designing PLP-dependent enzymes and could be of great importance to the design of other homodimeric enzymes as well.