There are two trends in current microbiological research. First, researchers have increasingly realized that intestinal microbes, especially a large number of anaerobic bacteria, are closely related to human health with the development of intestinal microbe-related research. Because the intestine itself belongs to an anaerobic environment to a certain extent, the cell physiology research of intestinal bacteria needs to be based on the anaerobic culture environment. Second, it is difficult to meet the requirements of cell heterogeneity only by relying on classical microbial population culture methods. Research on heterogeneity requires the development of methods to study bacterial physiology at the single-cell level for in-depth study of the physiological laws that are hidden by cell populations and ignored by researchers. Here, a method is developed for culturing bacteria under anaerobic conditions, including the design of related culturing equipment and corresponding experimental procedures. Based on stably maintaining the strict anaerobic condition of the culture environment, the microfluidic chip is used to long-term singlecell culture. Combined with high-resolution microscope time-lapse imaging technology, the real-time observation and data collection of growth dynamics of single bacterial cell can be realized. This method provides powerful technical support for single-cell analysis of bacterial cells under anaerobic conditions.