Oreochromis niloticus (Nile Tilapia), has the advantages of rapid growth, versatile feeding habits, the absence of intermuscular thorns, and robust stress resistance. Moreover, it is abundant in essential amino acids and unsaturated fatty acids, making it highly nutritious. Since 1976, the Food and Agriculture Organization of the United Nations has recognized Tilapia as a suitable fish for global cultivation. The farming model for Nile Tilapia in China is gradually shifting from traditional flowing water systems to high-density factory farming systems. However, oxygen deficiency is a common factor that leads to fish mortality in high-density intensive farming settings. Therefore, close attention must be paid to the dissolved oxygen (DO) status of aquaculture environments. DO is essential for maintaining normal metabolic activity in aquatic organisms and significantly impacts their growth, immunity, and energy metabolism. Studies have shown that fish can cope with low-oxygen environments through a series of physiological mechanisms. Under hypoxic conditions, fish utilize stored energy reserves (carbohydrates and lipids) to maintain their activities. Owing to the low utilization rate of carbohydrates in fish, utilizing lipids to provide energy is the optimal choice for them to adapt to low-oxygen environments. Owing to the low utilization rate of carbohydrates in fish, utilizing lipids to provide energy is the optimal choice for them to adapt to low-oxygen environments. Tilapia subjected to prolonged hypoxia utilized more lipids in response to hypoxic stress, suggesting that diets supplemented with appropriate lipid levels can enhance fish survival. However, the precise underlying mechanism remains unclear. This study aimed to investigate the interactive effects of DO content and fat levels on the growth performance, hepatic antioxidant capacity, immune parameters, and liver tissue structure of Nile Tilapia. The experiment was conducted using Tilapia with an initial weight of (7.62±0.29) g as the subjects. Two DO contents: hypoxia [(2.0±0.1) mg/L, group A] and normoxia [(5.0±0.1) mg/L, group B] and five fat levels (groups 1–5: 1.57%, 4.41%, 7.61%, 10.51%, and 13.01%) were applied, including three replicates per group. The culture period was 60 days. The results showed that as fat levels increased, the weight gain rate (WGR), specific growth rate (SGR), and feed efficiency of groups A and B first increased and then decreased, reaching maximum values in groups A2 and B3. At the same fat level, WGR and SGR were significantly higher in Group B than in Group A. Hepatic enzyme activities and content of various parameters, including catalase (CAT), total antioxidant capacity (T-AOC), aspartate transaminase (AST), cytochrome c oxidase (CCO), caspase-9, heat shock protein-90, malondialdehyde (MDA), hexokinase (HK), pyruvate kinase (PK), triglycerides, and hepatic lipase, were significantly affected by the interaction between oxygen levels and fat levels, whereas there was no interaction effects on adenosine triphosphatase (ATPase) activity between the two factors. With the increase in lipid levels, the CAT activity and T-AOC in group B and T-AOC in group A first increased and then decreased; the T-AOC in group A was lower than that in group B, and the B3CAT activity was significantly higher than that in the other groups. MDA content in group B increased gradually with the increase in fat level, that in group A decreased first and then increased, and that in group A was higher than that in group B. Under the same lipid level, the CCO and ATPase activities in group B were significantly lower than those in hypoxia group A. AST activity, T-AOC, and MDA content in group A were higher than those in group B, whereas caspase9 activities and HK, PK, and HSP90 contents in group B were significantly lower. Immune indicators, including immunoglobulin M, interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta, were significantly lower in group A compared to Group B and were not affected by fat level. DO and dietary fat levels significantly affected complement C3 in the liver of Tilapia. In groups A and C3, the content first decreased and then increased with an increase in fat level and was significantly lower in groups A3 and A4 than in the other groups. With increasing fat levels, the number of hepatic lipid vacuoles gradually increased in group B, whereas the cell morphology in group A2 was normal. Groups A3, A4, and B5 showed numerous lipid vacuoles, nuclear dissolution, and displacement. In summary, low oxygen levels and excessive dietary lipid levels lead to severe liver oxidative damage, fat accumulation, dysfunction of lipid metabolism, and reduced immune capacity. Within the scope of this experiment, feeding a diet with a fat content of approximately 4.41% during low oxygen conditions promoted the growth of Nile Tilapia and alleviated hypoxic stress, whereas feeding a diet with a fat content of approximately 7.61% in normoxic environments benefited the growth of Nile Tilapia.