In order to further exploit the by-products of Isatis indigotica Fort., the seed oil was studied for its extraction and physicochemical properties. Ultrasound-assisted extraction (UAE) was used, and the parameters affecting seed oil recovery were optimized through response surface methodology (RSM). The optimum conditions were as follows: solvent-to-sample, 24:1; particle size, 110 meshes; extraction temperature, 49 °C; and extraction time, 44 min. Which resulted in a maximum oil recovery of 81.20 ± 0.21% ( n = 3). Furthermore, the effects of UAE on the yield, fatty acid compositions, physicochemical properties, and microstructure of the seed powder were also investigated by calculating the recovery rate, utilizing a gas chromatograph fitted with a mass spectrometer (GC–MS), and performing scanning electron microscopy, respectively. The results show that UAE was an effective method for the seed oil extraction and the high content of unsaturated fatty acids (93.81%) demonstrates the oil has potential benefits for the cosmetics, edible products, or pharmaceutical industries.
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