Response surface methodology(RSM) was selected to optimize the fish oil nanoliposomes prepare by ethanol injection-dynamic high pressure microfluidizer(DHPM) method,its physical and chemical properties were evaluated at last.The results indicated that the optimum prepation technique parameters were as follows: phosphatidylcholine content was 29 mg/mL,the mass ratio of phosphatidylcholine,fish oil,cholesterol and Tween-80 was 10∶ 2∶ 2.5∶ 1,DHPM treatment pressure was 150 MPa and treated twice.Under these conditions,encapsulation efficiency(EE) of fish oil nanoliposomes was reached to 76.9%,average diameter was 128.1 nm,zeta potential was-20.11 mV.Fish oil nanoliposomes made by ethanol injection-DHPM exhibited great advantages in their characteristics,including small average diameter and narrow size distribution(polydispersity index 0.258) as well as great encapsulation efficiency and good stability.