Abstract: To explore an effective solution for high-value utilization of fish viscera, ultrasound coupled with subcritical water extraction (USCWE) was used to separate fish oil from Spanish mackerel viscera. The results were compared with those from enzymatic extraction (EE), ultrasonic-assisted organic solvent extraction (UOSE), and supercritical carbon dioxide extraction (SCE). Gas chromatography-mass spectrometry (GC-MS) was applied to analyze the fatty acid composition of the fish oil. The conversion rate of hydrothermal liquefaction (CRHL) of Spanish mackerel visceral stroma protein (SMVSP) was determined to explore the chemical mechanism of USCWE. Scanning electron microscope (SEM) was applied to observe the destructive action of ultrasonic physical effect coupled with subcritical water hydrothermal liquefaction on the network structure composed of stroma protein. The optimal conditions for USCWE of fish oil were consistent with that for the CRHL of SMVSP. The maximum extraction yield (EY, 59.87%±2.86%) of fish oil and CRHL of SMVSP (92.37%±3.12%) were obtained at 260 ℃ and 10 MPa with ultrasound enhancement (250 W/L, 20 kHz) for 60 min. Compared with the other three extraction methods, the EY of fish oil by USCWE was the highest. Moreover, the contents of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil were also the highest, which were (46.01±0.31) mg/g and (116.78±0.91) mg/g, respectively. The fish oil obtained by SCE showed the lowest acid value (AV, (7.15±0.33) mg KOH/g), peroxide value (POV, (1.83±0.13) mmol/kg), and carbonyl group value (CGV, (2.01±0.21) mEq/kg). USCWE could effectively destruct the network structure composed of stroma protein and fully released the fish oil wrapped in the mesh which had the advantages of environmental protection, short extraction time, high extraction yield and high content of EPA or DHA. The oxidation stability of fish oil by SCE was the highest, but the extraction time of SCE was longer and the EY was lower. This study elucidated the mechanism of USCWE of fish oil from Spanish mackerel viscera and provided experimental and theoretical basis for the precise use of this technology.