This article preliminarily discusses the effects of perfluorooctane sulfonate (PFOS) on the acute lethality and metabolism of Macrobrachium rosenbergii juveniles using a hydrostatic toxicity experiment and non-targeted metabonomics. Additionally, a chronic toxicity experiment was used to analyze changes to antioxidant enzyme activities in the gills, hepatopancreas, and gastrointestinal tract of juvenile shrimp under PFOS stress (0.1, 1, 5 ng/mL). The results showed that the 96 h half-lethal concentration of PFOS stress on Macrobrachium rosenbergii was (0.68±0.22) mg/L, and the safe concentration was 0.068 mg/L. Following 24 h of PFOS stress (20 ng/mL), 30 metabolic compounds with significant differences were identified in the gills of juvenile shrimps, 19 significantly differential metabolic compounds were identified in the hepatopancreas, and 24 metabolic compounds with significant differences were identified in the gastrointestinal tract. These metabolic compounds with significant differences were involved amino acid metabolism, fatty acid metabolism, and phospholipid metabolism. Under chronic PFOS stress, different exposure times and concentrations of PFOS may affect the activities of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and acid phosphatase (ACP) of Macrobrachium rosenbergii. There were also differences in the effects of phosphatase (ACP) activity, the activities of SOD, CAT, and ACP were inhibited, and the MDA content increased with prolonged exposure time. In summary, it is speculated that PFOS can cause oxidative damage to the gills, hepatopancreas, and gastrointestinal tissues of juvenile shrimp, it also affects the physiological metabolism of Macrobrachium rosenbergii. The results provide basic data and a reference to explore the toxic effects of PFOS on aquatic organisms.