Microalgae are considered a rich source of high-value metabolites with an array of nutraceutical and pharmaceutical applications. Different strategies have been developed for cultivating microalgae at large-scale photobioreactors but high cost and low productivity are the major hurdles. Optimizing the composition of media for the cultivation of microalgae to induce biomass production and high-value metabolite accumulation has been considered as an important factor for sustainable product development. In this study, the effect of plant growth regulators together with basal microalgal cultivation medium on biomass, total lipid, and EPA production was studied using the Plackett–Burman model and Response surface methodology. The traditional one-factor-at-a-time optimization approach is laborious, time-consuming, and requires more experiments which makes the process and analysis more difficult. The Designed PB model was found to be significant for biomass (396 mg/L), lipid (254 mg/L), and EPA (5.6%) production with a P value < 0.05. The major objective of this study is to formulate a medium for EPA production without compromising the growth properties. Further, we had formulated a new media using RSM to achieve the goal and the significant variables selected were NaNO3, NaH2PO4, and IAA and was found to be significant with 16.72% EPA production with a biomass production of 893 mg/L with a P value < 0.05. The formulated medium can be used in large-scale cultivation systems which can enhance biomass production as well as the omega 3 fatty acid production in marine microalgae Nannochloropsis oceanica.Supplementary InformationThe online version contains supplementary material available at 10.1007/s43393-021-00069-1.