Thraustochytrium sp. (T18) is a marine heterokont that exhibits a fast growth rate and accumulates a high proportion of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), as essential compounds for health. The extraction of DHA-rich oil from marine microorganisms is challenging due to the difficulty in the substantial cost of lyophilization, the incompatibility of solvents with wet biomass, and the large volumes of organic solvents required by traditional extraction techniques. To address these concerns, ionic liquids (ILs) have been used to improve the oil yield from a wet oleaginous microorganism under mild conditions. In this study, a novel hydrophobic IL, 1-decyl-3-methyl-imidazolium bis(2,4,4-trimethylpentyl)phosphinate ([C10mim][( i C8)2PO2]) was first applied as pretreatment to aid in the extraction of DHA-rich oil from T18 biomass in the aqueous phase. A central composite design and response surface methodology were used to study the effect of different extraction variables on the oil yield (temperature, pH, and the mass ratio of IL to dried T18). The extraction conditions were numerically optimized to maximize the oil yield [69.4 ± 0.5% (w/w)] within the experimental range (the temperature of 76 °C, pH of 5.0, and mass ratio of 4.2). In addition, the extraction efficiency was up to 97.4 ± 0.4% of the total lipid content. The extracted oil contained 44.7 ± 0.2% DHA under 1 h of the pretreatment process. In this work, the proposed approach was found to be efficient and sustainable for the water-compatible recovery of a DHA-rich oil recovery process.