A feeding study with Two-banded Seabream Diplodus vulgaris was conducted to determine the effects of replacement of fish oil (FO) by unrefined peanut oil (PO) on growth performance, feed utilization, body composition, fatty acid composition, and serum biochemical and hematological variables. Three isonitrogenous (35.8%) and isoenergetic (21.15 kJ/g) diets were formulated by replacing dietary FO with PO at levels of level 0%(PO0), 50% (PO50), or 100% (PO100). Fish were fed twice a day until satiation for an experimental period of 8 weeks. The best growth performance was observed in fish fed with the PO0 and PO50 diets. A significant increase was observed in hematocrit and mean corpuscular volume levels of fish fed with PO50 diet compared with the other groups. Hemoglobin, mean corpuscular hemoglobin, total protein, albumin, and globulin were not affected by dietary PO treatment and did not differ among experimental groups. The glucose level was highest in the PO100 group. Triglyceride and cholesterol levels were lower in fish fed diets with PO inclusions than in those fed the control diet. The fatty acid composition of fish was significantly affected by the experimental diets. Glutamic oxaloacetic transaminase, glutamic pyruvictransaminase, lactate dehydrogenase, and alkaline phosphatase were not affected by dietary PO treatment. The n-3:n-6 ratio in fish fed the PO0 diet was also higher than in fish fed the PO-supplemented diets. The results of the present study showed that FO could be substituted by PO up to 50% in Two-banded Seabream diets without any negative effect on growth performance or serum biochemical and hematological features.