The invention relates to microbial oil and a preparation method thereof. The microbial oil has unsaturated fatty acid content greater than 30wt%, triglyceride content less than 90wt% and diglyceride content greater than or equal to 5wt% and less than or equal to 20wt%. The preparation method comprises 1, carrying out oleaginous microorganism fermentation to obtain PUFA microbial oil-rich fermentation broth, 2, collecting the PUFA microbial oil-rich microorganisms and carrying out extraction and filtration to obtain mixed oil, and 3, adding lipase and water into the mixed oil, carrying out enzymolysis and carrying out purification to obtain microbial oil, or adding a diglyceride-containing mixture into the mixed oil, carrying out uniform mixing and removing a solvent so that microbial oil is obtained. The microbial oil contains an appropriate amount of diglyceride and is conducive to formation of a stable emulsion from the microbial oil. In microcapsule preparation, microbial oil can be well coated so that microcapsule surface oil content is reduced, a microcapsule antioxidation capability is improved, a microcapsule shelf life is properly prolonged and later production and use are promoted.