Abstract: To address the issue of diarrhea in farmed animals and develop environmentally friendly, safe, and effective alternatives to antibiotics in feed additives, this study focused on Lactobacillus acidophilus LA18（GDMCC 62222）, a strain of lactic acid bacteria isolated and preserved in-house. Through specific culture media and fermentation conditions, a bacteriocin was isolated and purified from the fermentation broth. Its molecular weight was determined using high-resolution mass spectrometry, and its minimum inhibitory concentration（MIC）and antibacterial spectrum were evaluated using the microbroth dilution method and the agar diffusion method. Additionally, the stability of the bacteriocin under different pH and temperature conditions was investigated, and its hemolytic activity was tested to assess its biological safety. The results showed that the bacteriocin had a molecular weight of approximately 3320 Da and exhibited significant inhibitory activity against Staphylococcus aureus（MIC: 0.4 μg·mL⁻¹）, while showing no significant inhibitory effect on common probiotics such as Bacillus subtilis, Enterococcus faecium, and Lactobacillus plantarum. It maintained good stability under pH conditions＜8.0, but was easily inactivated in strong alkaline environments. It remained stable after treatment at 70℃, 80℃, and 90℃ for 10 minutes, with a hemolysis rate of less than 5%, indicating good environmental tolerance and biological safety. This study provides new insights into the development of novel feed additives and the improvement of animal gut health by systematically evaluating the biological functions and safety characteristics of the bacteriocin produced by Lactobacillus acidophilus LA18.