Objectives: To develop a cost-effective anaerobic culture method for intestinal bacteria using multi-layer liquid seal and chemical adsorption under aerobic conditions. Results: The proposed method successfully cultivated anaerobic bacteria, including Clostridium butyricum, Bifidobacterium longum, and Enterococcus faecalis, demonstrating its efficacy. Fructose and procyanidin were tested, showing positive effects on bacterial growth. Multi-day transfer cultures of mixed intestinal bacteria confirmed the method’s repeatability and reliability, enabling efficient in vitro growth detection of numerous intestinal bacteria using standard anaerobic media. Conclusions: A novel, simple anaerobic culture method under aerobic conditions enables cost-effective and reliable growth detection of intestinal bacteria for drug effect comparisons.