This study utilised response surface methodology to optimise the conditions for the extraction of A. rugosa seeds oil (ARO). Single-factor experiment and response surface methodology (RSM) were performed to identify the extraction time, liquid-solid ratio and extraction temperature that provided the highest yield of ARO. The optimal extraction time, liquid-solid ratio and extraction temperature were 8 h, 4:1 mL/g and 55 °C. The fatty acids (FAs) content and oil yield obtained through the optimised impregnation-extraction process were 19.67 mg/g and 32.1%. These values matched well with the predicted values. Linolenic acid was identified to be the main active ingredient of ARO. The high-performance liquid chromatography-charged aerosol detection method presented here is fast and does not require derivatisation. Therefore, it could be used to quantitatively analyse the FAs present in ARO and applied to detect compounds with low or no ultraviolet response.