Nowadays, Pangasius hypophthalmus fillet is one of the main marine products in Vietnamese Mekong River Delta with over three hundred thousand tons per year. One of the byproducts of this industry is fat, about one hundred thousand tons per year. That byproduct has been only used in animal foodstuff industry after oil extracting or it becomes waste. In order to increase the quality of Pangasius hypophthalmus oil, we have enriched PUFA in the oil by hydrolysis with immobilized Rhizopus oryzea lipase. Lipase has been immobilized on celite by absorption method. Immobilization experiments have been designed in a two-level full factorial model with three factors. The fish oil has been hydrolyzed with immobilized lipase to enrich PUFA with optimized parameters found out: the ratio of enzyme to support of 1:10 g/g, the ratio of phosphate buffer volume to support weight of 30.55 ml/g, incubation time of 5.15 hours. The result has showed that 60-minute hydrolysis is the most appropriate. Additionally, the oil iodine value has increased from 71.4 to 97.4, and DHA concentration has risen from 1156mg/kg to 1916mg/kg.