Abstract: In order to improve the utilization of Kosteletzkya virginica seeds and develop a new material for biodiesel, Kosteletzkya virginica seeds were taken as raw materials and the supercritical CO2 fluid extraction technology was used to extract seed oil. The single factor experiment and the orthogonal experiment were carried out to study the effects of factors such as extraction pressure, extraction temperature, extraction time and supercritical CO2 flow on oil yield. The optimal technological conditions of oil extraction from Kosteletzkya virginica seeds by supercritical CO2 fluid were determined. The order of factors that influenced the oil yield within the experimental range was as follows: extraction pressure＞extraction temperature＞supercritical CO2 flow>extraction time. The optimal technological parameters of oil extraction from Kosteletzkya virginica seeds should be as follows: extraction pressure 25MPa, extraction temperature 45℃, supercritical CO2 flow 21 kg/h and extraction time 100 min, and under such conditions, the total oil yield after extraction three times is 19.35%