Abstract: Two-step enzymatic hydrolysis was uesd to extract oil from Schizochytrium sp. and the extraction conditions were optimized.The optimal extraction processing of oil from Schizochytrium sp were explored by single factor and orthogonal experiments, and verification experiments was used to verify the result of orthogonal experiments. The optimal extraction conditions was obtained as follows: liquid/solid ratio 1 ∶ 7, neutral protease addition 9%, enzymatic hydrolysis temperature45 ℃, enzymatic hydrolysis time 3 h, enzymatic hydrolysis pH6.5, alkaline protease addition 12%, enzymatic hydrolysis temperature 70 ℃, enzymatic hydrolysis time 6 h, enzymatic hydrolysis pH9.5. Under these conditions, the clear oil extraction ratio from Schizochytrium sp. reached 90.22%. Gas chromatography-mass spectrometry ( GC-MS) analysis showed that thecontent of unsaturated fatty acids was 44.13% and the DHA content was 32.06%.