Full factorial experimental design was used to optimize the operating conditions of lactose converting into lactulose under electro-activation conditions. The ANOVA showed a high correlation coefficient R² (0.9889) and a low root mean square error (1.4 ± 0.15%) values for the regression model of the experimental design, indicating the good predictive nature of the model. The obtained results after optimisation showed that the optimum electro-isomerization conditions are the electric current of 300 mA, temperature of 10 °C, and reactor configuration in which a cation exchange membrane separated the central compartment from the cathodic one in which the electro-isomerization reaction was conducted. According to the optimized conditions, the maximum lactose conversion into lactulose was 30 ± 1.23%. As by-products of the electro-activation, only small amounts of galactose were observed.