Acutodesmus obliquus is widely known as oleaginous microalgae that is able to produce α-linolenic acid (ALA). In this study, this microalgae was detected to produce eicosatetraenoic acid (ETA) which is a very rare omega-3 fatty acid. Recently, there has been renewed interest in ETA which has shown to have a pivotal role in anti-inflammatory activities. ETA is of significance due to their diverse functions that are little investigated. The aim of the study was to enhance the yield of ALA and ETA produced by Acutodesmus obliquus CN01 by optimising various culture conditions using Taguchi method that are much simpler (minimum number of experiments) with time and cost-saving. Four factors studied were carbon, nitrogen, salinity, and phosphorus. Taguchi method was performed to evaluate the optimisation of omega-3 fatty acids production under different culture conditions. The results demonstrated that the optimal culture conditions can be obtained by following the combination of 0.5 gL-1 of sodium acetate, 0.015 gL-1 of sodium nitrate, 0.58 gL-1 of sodium chloride and 0.0002 gL-1 of dipotassium hydrogen phosphate concentration. Carbon is observed as the main factor contributing to omega-3 fatty acids production. All sets of experiments produced both ALA and ETA. The result suggested that Acutodesmus obliquus CN01 has shown to be potential omega-3 fatty acids producer including the rare ETA which is of beneficial to the pharmaceutical industry and also food industry.