n-3 PUFAs have various physiological activities， but their contents in natural oils are usually low. This study investigated the preparation of glycerides rich in n-3 PUFAs by Eversa Transform 2.0(ET 2.0)-catalyzed alcoholysis of fish oil. The results showed that the ET 2.0 from Aspergillus oryzae could effectively identify n-3 PUFAs during alcoholysis. Saturated fatty acids and monounsaturated fatty acids were released from fish oil by ET 2.0-catalyzed alcoholysis， causing the production of n-3 PUFA-enriched glycerides. Under optimal conditions (1.6 ∶ 1 of mass ratio of methanol to algal oil， 40% methanol aqueous solution， 4% ET 2.0 dosage， 35 ℃， 2 h)， the n-3 PUFA content in glyceride increased from the initial 39.88% to 67.87%， the content of EPA and DHA increased from 36.61% to 65.22%， and the yield of n-3 PUFAs was 56.33%. When tuna oil and algal oil were used as substrate， the content of n-3 PUFAs in tuna oil glycerides increased from 38.81% to 61.35%， and the content of n-3 PUFAs in algae oil glycerides increased from 45.96% rose to 64.09%. The n-3 PUFAs-enriched glycerides obtained by the above method have great application potential in the food and pharmaceutical industries.