Lactococcus lactis (L. Lactis) NZ9000 is one of the most widely used host for the food-grade efficient protein expression system. In order to obtain high electrotransformation efficiency of NZ9000, single factor experiment was carried out to optimize the factors effecting electrotransformation efficiency, such as cell growth, glycine content, plasmid content, electric field strength, resuscitation medium and recovery time. The above key factors were further optimized by using response surface methodology and the optimum values for optical density OD₆₀₀, glycine content and electric field strength were 0.2, 8 g/L and 12.5 kV/cm, respectively. The electrotransformation efficiency (3.76×10⁷ CFU/μg DNA) was increased by the present of 250 compared to that in the initial conditions. The optimized preparation method of L. lactis NZ9000 improves the electrotransformation efficiency, which lays a foundation for exploring the competence potential of L. lactis.