Robust and versatile promoters for Lactobacillus plantarum found in wine are necessary gene expression tools for genetic research involving wine stress. We optimized the electrotransformation parameters for L. plantarum XJ25 isolated from wine and engineered five promoters based on the promoter P23; these promoters showed significantly different transcriptional activities under nonstress conditions. The activities of these promoters in vivo and the resulting growth burden to the host strain under different wine stresses were also evaluated. A range of colors (from white to dark pink) of the developing colonies with the plasmid pNZ8148 carrying an X-mCherry expression cassette, namely, P23-mCherry, trcP23-mCherry, POL1-mCherry, POL2-mCherry, POL3-mCherry, or POL4-mCherry, were analyzed. The applicability of the optimized electrotransformation parameters and synthetic promoters with different activities were also verified in several L. plantarum strains. Therefore, the optimized electrotransformation and these characterized promoters were determined to be suitable for applications in wine research in the future.