Selenium (Se) is known as an essential nutrient for human health. More than 15% of world population suffers Se deficiency mainly due to consumption of poor Se food crops. Biofortification has been introduced as a sustainable and cost-effective approach to combat Se deficiency in the food chain. Onion as a high-consumed and low price vegetable has a high ability for Se uptake and conversion of this element to anti-carcinogenic compounds. Due to similar physical and chemical properties, Se and sulfur (S) compete with each other in uptake, translocation and accumulation in plant tissues. It has been known that amino acids as reduced source of nitrogen not only affect selenate and sulfate uptake, but reduce nitrate concentration in vegetables. This study investigated the effect of selenate, sulphate, and certain amino acids on yield and bulb Se and nitrate concentration with the aim of biofortification programs. A sand culture experiment was set up in a completely randomized factorial design with three replicates. Two onion cultivars ( Allium cepa L., cvs. Dorrcheh and Cebolla) were grown in nutrient solutions in which 20% of nitrate was replaced with arginine, histidine, or mixed amino acids extracted from blood powder and exposed to two levels of sulfate (1 and 3 mM) and two levels of selenate (0 and 25 uM). A control with no nitrate replacement was also considered. Blood powder sample was collected from Blood Powder factory of Fasaran that is located at 45 km north Isfahan. In this factory, animal blood is converted to powder at high pressure and temperature. Proteins of blood powder were extracted by using hot acid hydrolysis method. The amino acids were extracted from blood powder using the method of acidic hydrolysis of proteins. After plant harvesting, fresh and dry weight of bulb, concentration of nitrate, ammonium, total N, total amino acids, total S, and Se and activity of nitrate reductase (NR), and gluthamin synthetase (GS) were measured. Results obtained from the present study are presented in three parts. In the first part, interaction of Se and S on Se-enrichment of onion bulb was investigated. Results showed that addition of Se increased bulb yield of both cultivars. While Se application had no effect on sulfate uptake, increasing sulfate level in the nutrient solution decreased bulb Se concentration of Dorcheh (by 48.5 %) and Valensiana (by 45.0%) cultivars. Increasing sulfate concentration in the nutrient solution increased pyrovic acid concentration of both onion cultivars. Addition of Se, reduced concentration of pyrovic acid in Valensiana exposed to 1 mM sulfate treatment by 15%. In general, results showed that supplementation with Se and 3 mM sulfate improved yield and marketing and nutritional quality of Valensiana bulbs. It seems that production and consumption of Se-enriched onion bulbs can be used as a substitute for Selen Plus supplements that are the most common Se-supplements in the markets. In the second part, the effect of replacement of 20% nitrate-N in the nutrient solution by arginine, histidine, and blood powder amino acids on nitrate metabolism and bulb yield of two onion cultivars was investigated. Addition of amino acids into the nutrient solution, regardless of amino acid treatment, significantly reduced nitrate accumulation in onion bulb. This increase was accompanied with elevated activity of glutamine syntetase (GS) and higher production of ammonium in onion bulb. Results showed that nitrate reductase activity (NR) and ammonium concentration in plants supplied with amino acids were lower than those non-supplied with amino acids. The mixture of amino acids extracted from blood powder was more effective than sole arginine and histidine treatments in decreasing bulb nitrate concentration. Application of amino acids significantly enhanced fresh and dry matter yield of onion bulb although this increase was greater by blood powder-extracted amino acids in comparison with histidine and arginine treatments. According to the results obtained, the mixture of amino acids extracted from blood powder can be used as a source of nitrogen in nutrient solution culture of onion. In the third part, the effect of amino acids extracted from blood powder on Se and pyrovic acid concentration of two onion cultivars was investigated. Results showed that different amino acid treatments affected assimilation of selenate and sulfate by affecting their uptake from the nutrient solution. In both onion cultivars, the highest increase in Se and sulfate uptake was observed at the mixed blood powder-extracted amino acid treatment. Keywords : Biofortification, Selenium, Sulfate, Blood powder, Amino acid, Nitrate, Onion