Abstract: To prepare a probiotic fermented milk that can produce antibacterial substances in situ, Limosilactobacillus reuteri HLRE05, which has the ability to metabolize glycerol into broad-spectrum antimicrobial Reuterin, was used to investigate the optimum conditions for preparation of the fermented milk, to evaluate the effects of glycerol addition on the Reuterin production and the properties of the fermented milk, to analyze the biochemical indices and the Reuterin content of the fermented milk during storage, and to determine the antibacterial ability. The results showed that the optimal preparation conditions for the Reuterin-containing fermented milk was a total inoculum volume of 5%, with a 1:1 ratio of YO-MIX 495 to HLRE05, a sugar content of 6% (a 1:1 ratio of rhamnose to sucrose), and a fermentation temperature of 37 ℃. When supplemented with glycerol at a concentration of 100 mmol/L, the in situ production of Reuterin reached 0.28 mg/mL, and the content of L. reuteri and water retention in fermented milk were better. In addition, fermented milk supplemented with glycerol maintained a stable pH and was characterized by the effective inhibition of pathogen proliferation during storage. This study could pave the way for the exploration and application of characteristic lactic acid bacteria, such as L. reuteri HLRE05, in dairy products.