This study examined the effects of different water flow durations on serum biochemical indices, liver antioxidant capacity, and intestinal health of largemouth bass (Micropterus salmoides) in a recirculating aquaculture system (RAS). During a 90-day experiment, 270 largemouth bass (initial weight 96.05 ± 18.87 g) were randomly assigned to three groups: a control group (CG) with a constant flow velocity of 0.3 cm/s, an interval exercise group (IE) with a water flow velocity of 2 bl/s for 8 h/day, and a sustained exercise group (SE) with a continuous flow velocity of 2 bl/s for 24 h/day. The results showed that the IE group had higher total protein (TP) levels than the CG and SE groups (P < 0.05). In contrast, the SE group had lower triglyceride (TG) and cholesterol (CHO) levels (P < 0.05), indicating that exercise intensity significantly affects serum biochemical parameters. The study also evaluated the effects of sustained swimming on liver antioxidant capacity, digestive enzyme activity, intestinal health, and microbial community composition. Antioxidant assays revealed significantly increased activities of superoxide dismutase (T-SOD) and catalase (CAT) in the SE group, along with the lowest malondialdehyde (MDA) levels (P < 0.05), indicating enhanced antioxidant defenses. Enzyme assays showed a significant increase in trypsin and lipase activities due to swimming exercise (P < 0.05). Intestinal morphology analysis showed increased villus height and wall thickness in the SE group, without visible tissue damage, suggesting improved intestinal structural integrity. Gene expression analysis showed a significant upregulation of pro-inflammatory genes in the IE group and an increase in anti-inflammatory gene expression in the SE group. Microbial community analysis showed no significant differences in alpha diversity, species richness, or total diversity among the groups. However, variations in microbial species distribution at the phylum and genus level were observed, which were likely influenced by exercise intensity. In conclusion, sustained swimming exercise significantly improved antioxidant capacity, intestinal health, and modulated the immune response in largemouth bass. This highlights its potential to improve fish health and sustainability in RAS.