The present study aimed to evaluate the effects of dietary copper nanoparticles (Cu-NPs) and vitamin C (VC) supplementations on rainbow trout (Oncorhynchus mykiss) juveniles. Six trial diets were supplemented with Cu-NPs and VC including 0/0 (T1, control diet), 0/250 (T2), 0/500 (T3), 2/250 (T4), 2/500 (T5), and 2/0 (T6) mg Cu-NPs/VC per kg diet. After the feeding trial for 60 days, the fish were challenged with Yersinia ruckeri and the survival rate was calculated for 15 days. Based on the data analysis, weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR), protein efficiency ratio (PER), lysozyme, alternative complement activity (ACH50), catalase (CAT), glutathione peroxidase (GPX), hematocrit (Hct) and mean corpuscular volume (MCV) were significantly (p < 0.05) affected by the Cu-NPs factor. Meanwhile, VC was a significant factor for hemoglobin (Hb) and superoxide dismutase (SOD) (p < 0.05). The results showed that the Cu-NPs and/or VC-supplemented diets improved WG, FCR, SGR, PER, lysozyme, ACH50, SOD, CAT, GPX, Hb, Hct, and MCV when compared with the control group (p < 0.05). The expressions of TNF-α, IL-1ß, IL-10, SOD, CAT, and GPX genes were significantly (p < 0.05) decreased in the fish fed on T3, T4, and T5 diets versus the control. In addition, the dietary Cu-NPs and VC supplementations significantly enhanced resistance against pathogens and led to the control of infection in rainbow trout. In conclusion, Cu-NPs and VC administered as feed additive at 2/250–500 mg/kg elevated the growth performance, antioxidant capacity, and health of rainbow trout.