Abstract: Perfluorooctane sulfonate (PFOS), as the main active component in anti-fouling agents used for textile and leather products, has been widely used in both industrial and daily settings. So far, PFOS contamination has been detected in groundwater, surface water, seawater, and even animal tissues. It exhibits notable toxicity to the liver, reproduction, development, and nervous system of fish; however, the effect on the fish intestine remain unclear. In this study, male and female zebrafish were exposed to 0, 1, and 10 mg/L of PFOS for 21d, respectively, and the changes in intestinal morphology, structure, and microflora of zebrafish were detected by survival rate, intestinal tissue section, 16S rRNA high-throughput sequencing, real-time quantitative PCR, and other techniques. The results showed that zebrafish exposed to 10 mg/L PFOS had a significantly higher mortality rate than those exposed to 1 mg/L, with males being more affected than females. The survival rates of male and female fish in the 1 mg/L concentration group were 93.3% and 83.3%, while those in the 10 mg/L concentration group were 33.3% and 13.3%, respectively. Exposure to 1 mg/L PFOS led to a reduction in intestinal thickness and damage to intestinal villi. In the 10 mg/L group, the intestine was significantly thinner, villi height was reduced, and intestinal mucosal epithelial cells were swollen and accompanied by severe dissolution. After 21d PFOS exposure, the inflammation-related genes such as tumour necrosis factor-alpha (TNF-α), interleukin1beta (IL-1β), and interleukin10 (IL-10), along with caspase3, p53, and B-cell lymphoma-2 (Bcl2), were significantly higher than those of the control group. Furthermore, caspase3 and p53 expression in male intestines was significantly higher than those in females (P<0.05). PFOS exposure also significantly increased the diversity and altered the structure of gut microbiota, with a marked rise in the relative abundance of Firmicutes, Proteobacteria, Actinobacteriota, and Bacteroidota, and a significant decline in Fusobacteria. At the genus level, PFOS treatment increased the relative abundance of Ralstonia and Pseudomonas, but decreased Cetobacterium and Aeromonas. The Firmicutes/Bacteroidetes (F/B) ratio was higher in both treatment groups compared to the control group. Additionally, PFOS treatment increased the amino acid and lipid metabolism of the gut flora, but decreased the synthesis and metabolism of glycans. In conclusion, long-term PFOS exposure can lead to structure damage to the intestinal and dysbiosis in the microbial flora of aquatic animals, subsequently affecting their health.