A new process for subcritical extraction of Cucumariafrondosa oil was established using freeze-dried Cucumariafrondosa visceral which is rich in lipid active substances. The subcritical extraction conditions were optimized by single factor and response surface methodology, and the best extraction process was obtained: material-liquid ratio 1∶3 （g/mL）, extraction pressure 0.4 MPa, extraction times 5 times, extraction time 56.18 min, extraction temperature 48.55 ℃, extraction rate 23.38%. By comparing the physical and chemical indexes of oil extracted by different methods and gas chromatography-mass spectrometry (GC-MS) graphs, the physicochemical indexes of oil extracted by subcritical method are better than other methods, the content of EPA in the oil reaches 40.7%, indicating that the subcritical method for extracting Cucumariafrondosa oil has high process efficiency and more stable oil quality.