Antibiotic resistance genes are widely used to produce plasmid DNA vaccines, but risk unwanted exposure to antibiotic residues and the spread of resistance genes. To overcome the limitations of existing selection technologies, we developed an alternative system applying the widely used household biocide triclosan as the selective agent and an endogenous growth essential target gene, fabI, as the plasmid-borne marker in Escherichia coli. The fabI/triclosan system enables efficient, non-antibiotic selection of transformed bacteria, with improved safety and plasmid production features. Here we aimed to evaluate the performance of this non-antibiotic selection system using a plasmid DNA vaccine against bovine viral diarrhoea virus as an example. The new system displayed high-yield plasmid DNA production in a standard E. coli host strain and growth media. Notably, the purified pDNA provided efficient in vitro protein expression and a strong in vivo neutralising antibody response in a mouse model, with measures comparable to that of the parental plasmid DNA based on ampicillin resistance. The fabI/triclosan system requires only low levels of triclosan for selection (1 μM) and residual triclosan in isolated DNA was below the limit of detection (< 20 parts per trillion). The fabI/triclosan selection system provides a simple, non-antibiotic resistance marker for plasmid selection, applicable to DNA vaccines and possibly other recombinant vaccine applications.