ETHNOPHARMACOLOGICAL RELEVANCE: As digestive health issues rise and interest in natural therapies grows, traditional herbs like Cassia Seed are gaining attention for their antioxidant, laxative, and digestive benefits. AIM OF THE STUDY: This study aimed to optimize the fermentation conditions of Cassia seed using microbial technology to enhance the content of anthraquinone compounds, thereby augmenting its pharmacological effects, particularly in promoting intestinal peristalsis and alleviating constipation. MATERIALS AND METHODS: Fermentation of Cassia Seed was conducted under controlled microbial conditions. High-performance liquid chromatography (HPLC) was employed to analyze the anthraquinone profile of the fermented solution. Mouse model was utilized to evaluate biochemical markers and intestinal functions, while in the zebrafish constipation model, calcein was utilized as a marker to monitor intestinal function and excretion dynamics. Cassia Seed was fermented under controlled microbial conditions. HPLC was employed to analyze the anthraquinone profile of the fermented solution. Mouse models were utilized to evaluate biochemical markers and intestinal functions, while a zebrafish constipation model used calcein as a marker to monitor intestinal function and excretion dynamics. RESULTS: The optimized fermentation significantly increased the content of anthraquinone compounds, including substances like emodin, aloe-emodin, rhein, chrysophanol, and physcion, as confirmed by HPLC analysis. In a mouse constipation model, the fermented solution reduced nitric oxide (NO) levels while increasing acetylcholine (Ach) and gastrin (Gas) levels, thereby promoting intestinal peristalsis and accelerating bowel movements. Additionally, the fermented solution facilitated repair of the colonic mucosa in mice without significant pathological damage such as degeneration or fibrous tissue proliferation. In the zebrafish model, the fermented Cassia Seed solution reduced calcein fluorescence intensity, indicating enhanced intestinal peristalsis and accelerated bowel movements, thereby decreasing calcein retention time in the body. CONCLUSION: This study investigated the potential clinical applications of fermented Cassia seed ferment in digestive health, providing a theoretical basis and inspiration for further research and development. Additionally, it offers new insights and possibilities for the development of novel natural medicines or functional foods aimed at improving constipation and promoting intestinal health.