Aquaculture feed formulation has recently turned its focus to reduce the reliance on marine-derived resources and utilise alternative feedstuffs, as an approach to improve the environmental sustainability of the aquaculture sector. The fish oil market is highly volatile, and availability of this commodity is continuously decreasing for use in aquaculture. Currently, a growing number of commercial efforts producing microalgae are providing omega 3-rich oil for sustainable aquaculture feed. This study was focused to determine the nutrient digestibility of a marine microalga, Schizochytrium spp., which is rich in docosahexaenoic acid (DHA) and long-chain polyunsaturated fatty acids (LC-PUFA), as a novel dietary lipid source that could be utilized effectively by rainbow trout (Oncorhynchus mykiss). A whole-cell Schizochytrium spp. biomass was used in the digestibility experiment at two different temperatures, 8 °C and 15 °C. No significant differences were detected between the two temperatures for the apparent digestibility coefficients (ADCs) of the dry matter (94.3 ± 4.9%), total lipids (85.8 ± 0.0%), crude proteins (89.5 ± 1.8%), energy (83.1 ± 1.7%) and fatty acids (85.8 ± 7.5%). The ADCs of the nutrients, energy, DHA and other fatty acids showed that Schizochytrium spp. is a high-quality candidate for fish oil substitution and supplement of LC-PUFA in fish feed with vegetable oils.