Abstract: In order to test the effect of galactooligosaccharide (GOS) on the characterization and antibacterial activity of milk fermented by Lactobacillus plantarum, the main factors affecting the fermented milk were investigated and the most suitable fermentation conditions for improving the characterization of fermented milk were obtained with response surface analysis. The enterotoxigenic Bacillus cereus HN001 was used as an indicator to study the effect of GOS addition on the antibacterial activity of L. plantarum ZDY2013 fermented milk. The results showed that L. plantarum could use GOS for metabolism in vitro and inhibit the growth of B. cereus effectively. The proper addition of GOS in milk could increase the number of viable bacteria, reduce the pH and enhance the water holding capacity of L. plantarum fermented milk. The result from response surface analysis was found that the best fermentation conditions for fermented milk were 2.0% inoculums of L. plantarum, 1.0% GOS, fermented at 42 ℃ for 24 h. Additionally, GOS addition in fermented milk could effectively control the concentration of enterotoxigenic B. cereus HN001 to below 10⁶ CFU/mL. The results of this study provide the foundation for the application of GOS and L. plantarum ZDY2013 in fermented milk.