The interactive effects of dissolved oxygen and feeding rates on Jinhu grouper in a recirculating aquaculture system. Four treatment groups were established: the Control group (CK) with a dissolved oxygen level of 6 mg/L and a feeding rate of 2%; the High Feeding Rate group (HFR) with a dissolved oxygen level of 6 mg/L and a feeding rate of 3.5%; the High Dissolved Oxygen group (HDO) with a dissolved oxygen level of 9 mg/L and a feeding rate of 2%; and the Mixed group (MIX) with a dissolved oxygen level of 9 mg/L and a feeding rate of 3.5%. A practical aquaculture experiment was conducted over a period of 60 days. Through the analysis of the intestinal microbiota composition and serum metabolomics of juvenile Jinhu grouper, a weighted correlation network analysis (WGCNA) was employed to investigate the associations between metabolomics and gut microbial genera. The results indicated that the HFR group significantly enhanced both species diversity and abundance of the gut microbiota. Meanwhile, the HDO group maintained abundance while optimizing the evenness of functional structure distribution. The MIX group achieved intestinal homeostasis by moderately upregulating diversity to prevent excessive competition among microbial communities. Metabolomics analysis indicates that both the HDO and HFR groups optimize the primary bile acid synthesis pathway by reducing the abundance of genera such as Cetobacterium and Stenotrophomonas, while simultaneously decreasing the abundance of Pseudomonas to modulate sphingolipid levels and alleviate immune stress. Notably, the MIX group effectively inhibits the proliferation of Vibrio under hyperoxic conditions and enhances serum spermidine levels by regulating the abundances of Acinetobacter, Dubosiella, and Allobaculum, thereby improving the antioxidant capacity. The conclusion indicates that the combined effect of a 3.5% feeding rates and 9 mg/L dissolved oxygen can optimize the intestinal microbiota structure of grouper, inhibit the proliferation of pathogenic bacteria, and enhance the functional activity of beneficial bacteria. The findings provide a theoretical basis for regulating dissolved oxygen levels and feeding parameters in RAS. It is recommended to adopt a combination strategy of a 3.5% feeding rates with 9 mg/L dissolved oxygen for aquaculture practices.