Abstract: The antibacterial effect of extraction from stevia rebaudiana was studied. Punch method was used to make bacterial susceptibility test for the extraction from stevia rebaudiana, and double-dilution method was used to test minimum inhibitory concentration. Besides, the influence of pH, ultraviolet radiation and temperature on the antibacterial effect of the extraction was also studied. The result showed that the extraction's minimum concentration to inhibit Escherichia coli, Staphylococcus aureus and Bacillus subtilis were 8.4, 8.4 and 4.2mg/mL respectively with the stronger antibacterial effect than potassium sorbate. pH3⁵ was suitable for the extraction to inhibit bacteria. The extraction from stevia rebaudiana showed loss of antibacterial effect to Escherichia coli after treated with ultraviolet while ultraviolet had little effect to Staphylococcus aureus and Bacillus subtilis.Atimicrobial activity was not changed obviously by heating at 121℃ for 30min. It showed that extraction from stevia rebaudiana was a novel, nature preservative.