Microbial adaptation to environmental stress involves complex adaptations of bacteria. Many such responses are transient and dynamic. However, monitoring the dynamic responses of live bacteria to stimulations at the molecular level remain a challenge. This work describes the development of syringe spray mass spectrometry (MS) method that allows direct analyses of molecules released by the bacteria in responses to external stimuli with second level time resolution. We report the application of this technique to visualize the dynamic release of small molecules from Escherichia coli ( E. coli) under ethanol and isopropanol treatments. With the unique time-resolved capability, detailed destruction process of alcohol on bacteria cell wall could be observed. Compared to other ethanol concentrations, 75% ethanol showed stronger damages to lipopolysaccharide (LPS) and peptidoglycan located on E. coli cell wall. Furthermore, isopropanol showed stronger liposolubility and permeability, and an equilibrium could be achieved in a much shorter time.