Here, we investigated the effects of total dietary fish oil (FO) substitution with plant oil (PO) on hematological indices, immune status, antioxidant activity, IL1β and TNF-α gene expression, and hypoxia stress resistance in Oreochromis niloticus at suboptimal temperatures. Fish (n = 360)were randomly divided into 12 circular fiberglass tanks (500 L; 3 replicates for each dietary group, 30 fish/replicate, 90 fish/group). The control group was fed a basal diet with FO as the lipid source. The CO, SFO, and LSO groups were fed a basal diet with complete replacement of FO with corn, sunflower, and linseed oils, respectively. After 4 weeks, no effects on hematological indices were observed in fish fed the experimental diets. The LSO and CO groups showed a significant increase in γ globulin levels. The highest levels of non-specific immune parameters, antioxidant activity, and IL-1β and TNF-α gene expressions were recorded in the LSO group. The LSO group also exhibited good resistance to hypoxia stress. Therefore, the total dietary substitution of FO with PO (especially LSO and CO) is recommended as a valuable strategy to ameliorate the immunosuppressive effects of suboptimal temperatures and enhance the resistance of O. niloticus to hypoxia stress.