Objective To solve the bottleneck of plasmid instability during microbial fermentation of l-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. Results The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for l-DOPA biosynthesis. Conclusions The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for l-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.