Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of L-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, L-dihydroxyphenylalanine (L-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for L-DOPA production. In this study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was successfully overexpressed in Escherichia coli and screened for L-DOPA synthesis with a specific activity of 2.69 U mg(-1). Fn-TPL was found to be a tetramer, and the optimal temperature and pH for alpha, beta-elimination of L-tyrosine was 60 degrees C and pH 8.5, respectively. The enzyme showed broad substrate specificity toward natural and synthetic L-amino acids. Kinetic analysis suggested that the k(cat)/K-m value for L-tyrosine decomposition was much higher than that for L-DOPA decomposition, while Fn-TPL exhibited similar catalytic efficiency for synthesis of L-tyrosine and L-DOPA. With whole cells of recombinant E.coli as biocatalyst, L-DOPA yield reached 110 g L-1 with a pyrocatechol conversion of 95%, which was comparable to the reported highest level. The results demonstrated the great potential of Fn-TPL for industrial production of L-DOPA.