Docosahexaenoic acid (DHA) is the key omega-3 fatty acid for the growth and development of the human brain and retina and is inevitable for heart health. DHA is highly susceptible to environmental factors due to its increased oxidation and physical instability; therefore, preventing and extending its shelf life is highly required. DHA can be made stable in terms of its chemical and kinetic attributes with the help of nanoemulsion systems. In this study, a DHA nanoemulsion was prepared via an oil-in-water emulsion using Tween-40 emulsifier, through high speed homogenization (HSH), high pressure homogenization (HPH) and combination of the HSH + HPH techniques. The stability of the nanoemulsion was investigated using physiochemical methods under different storage conditions. The fabricated nanoemulsions were less than 88 nm in size with globular droplets having higher negative charges of −31.1 and −30.2 mV, which were produced by the combination and HPH technique, respectively. Higher Newtonian flow behavior was observed for the combination and HPH technique. The functional groups of DHA remained intact without undergoing any changes and functional activity during the nanoemulsification process, which was evident from the unchanged fatty acids profile. Similarly, there were no structural changes observed in all the DHA emulsions. The refrigerated (4 ± 1 °C) emulsion exhibited lower lipid oxidation than those stored at the temperature of 28 ± 1 °C and 40 ± 0.2 °C. Combination of the homogenization techniques was found to result in better physiochemical properties and stability over a period of 100 days during storage than the other emulsification techniques for the encapsulation of DHA.