Abstract: The objective of the study was preliminarily explore the antibacterial activity and stability of Thamnolia subuliformis in vitro, the plate drilling method was used to determine the antibacterial activity of methanol extract of Thamnolia subuliformis against common pathogenic bacteria in vitro. The indicator bacteria of Staphylococcus aureus was used to determine the effects of factors such as different temperature, pH and UV irradiation time on antibacterial stability. Petroleum ether, ethyl acetate and n-butanol were used to extract the methanol extract, determine the polarity of its antibacterial active substances, and determine the content of usnic acid. The results showed that the methanol extract of Thamnolia subuliformis had good antibacterial effect on Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, Listeria stephensi, Staphylococcus surface, Listeria evansi, Salmonella paratyphi A, Salmonella paratyphi B of Gram-negative bacteria and Candida albicans. The minimum inhibitory concentration and minimum bactericidal concentration against Staphylococcus aureus were 0.625 and 10 mg/mL respectively; The results of antibacterial stability test showed that temperature had no significant effect on the antibacterial activity of methanol extract of Thamnolia subuliformis (P>0.05), and it still had high antibacterial activity after treatment at 100 ℃ for 30 min; The antibacterial activity of UV irradiation for 40 min and 50 min decreased significantly (P<0.05), but the antibacterial rate remained above 90%; pH had the greatest effect on the antibacterial stability of methanol extract of Thamnolia subuliformis. When the pH was close to physiological neutral (pH6.0), its antibacterial activity was not significantly different from that of the control group (pH4.83)（P>0.05）, showing high antibacterial activity. When the pH was high (8.0, 10.0) or low (2.0, 4.0), the antibacterial activity of the treatment group decreased significantly (P<0.05), and decreased with the increase or decrease of pH, The content of usnic acid in methanol extract and ethyl acetate extract of Thamnolia subuliformis was 0.8613% and 0.9379%, respectively. The results showed that the methanol extract of Thamnolia subuliformis had a certain broad-spectrum antibacterial effect, low concentration and good stability. It can be used for the development and utilization of natural food preservatives and plant-derived antibacterial drugs.