Dissolved oxygen (DO) is essential for the survival of aquatic organisms, while its absence from water effectively compromises the health of fish in aquacultures. Here, we simulated acute hypoxia (DO: 1.14 ± 0.04 mg L−1), and reoxygenation (DO: 6.90 ± 0.14 mg L−1) conditions, analyzed tissue biochemical indicators, antioxidant parameters, and hypoxia signaling molecules in the brain, liver, and gills of yellow catfish exposed to various durations of deoxygenation (40 and 70 min), hypoxia (1, 3, and 6 h) and reoxygenation (1, 3, and 6 h). All enzymatic activities and gene expression in tissues followed a similar pattern throughout the experimental duration. During acute hypoxia and reoxygenation, the levels of 5-hydroxytryptophan (5-HT), cortisol (COR), and malondialdehyde (MDA) increased significantly in the liver and gills, however, this increase in the brain was primarily concentrated in the acute reoxygenation phase. Regarding antioxidant parameters, the activities of total superoxide dismutase (T-SOD) and catalase (CAT) increased significantly under acute hypoxia and exposure to reoxygenation, however, unlike in the brain and gills, the total antioxidant capacity (T-AOC) in the liver was significantly reduced under acute hypoxic exposure, indicating intertissue differences in the antioxidant capacity of yellow catfish. The mRNA expression of hypoxia signaling molecules (e.g. hif1a/2a) in the liver, gills, and brain was upregulated to varying degrees under acute stress, indicating that the hypoxia signaling pathway was activated in tissues of yellow catfish in response to low oxygen availability. Our research provides a valuable reference for further elucidating the response and adaptation mechanisms of fish to acute hypoxia and reoxygenation stress and may prove essential for avoiding oxidative damage and maintaining the quality of farmed fish.