To optimize the extraction process, the tilapia fish head oil was extracted by supercritical CO₂ extraction (SC-CO₂) from tilapia fish head. The signal factors were chosen by single-factor experiments and the optimal extraction conditions of tilapia fish oil by SC-CO₂ were optimized by response surface method. Then the quality and fatty acid composition of tilapia fish head oil were analyzed. The results showed that the optimum conditions were extraction pressure 36 MPa, extraction temperature 41℃ and extraction time 3.1 h, which was better than cooking method, alkali hydrolysis method and enzymatic method. The extraction rate under the optimal extraction condition by SC-CO₂ was 79.5%, and the iodine value, POV and acid value were 5.36±0.15 mg/kg, 4.27±0.33 mmol/kg and 139.0±8.9 g/100g respectively. The contents of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) were 27.85%, 41.74% and 30.41% respectively. The tilapia fish head oil was rich in fatty acid composition, which indicated excellent nutritional value and functionality.