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The seed oil of Microula sikkimensis had been intensively studied due to its pharmacological actions. In the present study, seed oil of Microula sikkimensis was extracted using supercritical fluid extraction (SFE). Determinations of the extracts composition were performed by gas chromatography (GC). An orthogonal array design (OAD), OA (3), was employed for optimization of the supercritical fluid extraction of the compound with regard to the various parameters. Four factors, namely pressure (21.0–27.0 MPa), the dynamic extraction time, temperature, and CO flow rate of the supercritical fluid, were studied and optimized by a three-level OAD. The effects of the parameters on the yield of seed oil were studied using analysis of variance (ANOVA). The results revealed that the pressure had a significant effect on the yield of seed oil ( p < 0.05), while the other three factors, i.e., CO flow rate, dynamic extraction time and temperature, were not identified as significant factors under the selected conditions based on ANOVA. The results show that the best values for the extraction condition of seed oil was pressure 24.0 MPa, extraction time 3 h, temperature 45 °C and a CO flow rate 20 L/h in the 20-L vessel. [ABSTRACT FROM AUTHOR]