Replacing dietary fish oil with DHA-rich microalgae Schizochytrium sp. and EPA-rich microalgae Nannochloropsis sp. for olive flounder (Paralichthys olivaceus) was examined. Three experimental isonitrogenous and isolipidic diets with lipid source provided by 50% fish oil (F50S50), 50% (M50F25S25) and 100% microalgae raw material (M100) respectively were compared with a soybean oil (S100) diet as control. Triplicate groups of olive flounder juveniles (16.5 AE 0.91 g) were fed the experimental diets, and a group was fed the control diets for 8 weeks in a recirculation system. Results showed feed efficiency and growth performance were not significantly changed when fish oil (FO) was totally substituted by soybean oil (SO) or microalgae raw material (MRM). The whole-body composition, lipid content of liver and muscle, and lipid composition of plasma were not significantly influenced by the total substitution of FO by MRM. The polyunsaturated fatty acids (PUFA) content of muscle and liver declined in fish fed S100 diet, whereas it was not significantly reduced in fish fed M50F25S25 and M100 diets. The total substitution of FO by MRM not only maintained the levels of arachidonic acid, EPA or DHA but also increased n-3/n-6 ratio. In conclusion, MRM as the sole lipid source is sufficient to obtain good feed efficiency, growth performance and human health value in olive flounder juveniles.