Selenium is an important micronutrient that has antioxidant, growth potential, and reproduction enhancement abilities in various organisms. The aquaculture industry is a significant contributor towards meeting the dietary requirements of a majority of the global population, which further warrants developing novel approaches for enhancing the production of dietary fish. This study was performed to assess the growth performance of Nile tilapia (Oreochromis niloticus) fingerlings (1 gm in average weight and 2.75 cm in average length) upon nano-selenium (Se-Nps) supplementation. Nanoselenium was synthesized using high-energy ball milling (HEBM) using a 10-hour dry milling technique at 10:1 ball-to-powder ratio (BPR), size characterized by XRD and TEM, followed by mixing with basal feed in desired concentrations (0.5 mg/kg, 1 mg/kg, and 2 mg/kg) and administration to Nile tilapia fingerlings for 30 days, followed by the evaluation of growth performance parameters, fatty acid profile analysis using GC-MS, and nutritional quality index (NQI): [Thrombogenicity Index (IT), Atherogenicity Index (IA), n-3/n-6, n-6/n-3)]. Nile tilapia supplemented with 1 mg/kg Se-Nps showed improved growth performance (RGR: 1576.04%, SGR: 4.70%, and FCR: 1.91), demonstrated by higher survivability (> 95%), isometric growth (coefficient of allometry, b = 2.81), and higher weight gain compared to control (RGR: 680.41%, SGR: 3.42%, and FCR: 1.31), 0.5 mg/kg Se-Nps (RGR: 770.83%, SGR: 3.61%, and FCR: 1.18) and 2 mg/kg Se-Nps (RGR: 383.67%, SGR: 2.63%, and FCR: 1.22). The average length-weight relationship assessed as the condition factor (K) was highest in the 1 mg/kg Se-Nps group compared to others (p < 0.05). GC-MS analysis revealed that Nile tilapia supplemented with 1 mg/kg Se-Nps showed better meat quality, higher amount of n-3 fatty acids, eicosapentaenoic acid, and docosahexaenoic acid, high PUFA/SAFA ratios (1.35) and n-3/n-6 (0.33) ratios, with low atherogenicity index (0.36) and thrombogenic index (0.44), and relatively low n-6/n-3 fatty acid ratio (3.00) compared to other groups. Overall, Se-Nps supplementation at 1 mg/kg enhanced the growth performance and meat quality in Nile tilapia, and therefore could be a potential growth-promoting micronutrient for aquaculture enhancement.