3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25 TPL mutants were predicted, and two were confirmed as exhibiting the highest L-DOPA production and named E313W and E313M. The L-DOPA production from E313W and E313M was 47.5 g/L and 62.1 g/L, which was 110.2% and 174.8% higher, respectively, than that observed from wild-type (WT) TPL. The K of E313W and E313M showed no apparent decrease, whereas the k of E313W and E313M improved by 45.5% and 36.4%, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity between for pyridoxal-5'-phosphate. Structural analysis of the mutants suggested increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield.