Abstract: Attenuated Salmonella VNP20009 specifically colonizes and proliferates within tumor tissues and inhibits tumor growth. It has been used as drug delivery vehicle or in combination with other therapies (such as chemotherapy), which shows a good application potential in tumor therapy. In this paper, study was conducted to determine the physiological changes of growth curve and formation of bacterial biofilm of VNP20009 under various environmental stresses, such as temperature, pH, and H₂O₂. The results showed that VNP20009 could grow normally under the conditions of 42 ℃, pH 6.5, and 1 mmol·L^-1 H₂O₂. Furthermore, the weak acid environment was beneficial to the biofilm formation of VNP20009. This study provides a basis for in-depth study of the survival mechanism and application of attenuated Salmonella.