OBJECTIVE The optimum conditions of SFE-CO2 extraction of Plantaginis Semen oil (SPO), the composition of SPO and its antioxidant activities of SPO were all investigated in this paper. METHOD Response surface method (RSM) was used to establish the mathematical model of SFE-CO2 extraction of SPO to obtain the optimum conditions based on Single factor experiments. Fatty acid compositions and contents of SPO were tested by GC-MS, and antioxidant activities of SPO were studied by DPPH and ABTS free radical elimination method. RESULT The optimum conditions obtained through RSM analysis were as follows: extraction tempreture 70 degrees C, extraction pressure 30 MPa, extraction time 120 min and flow rate 30 L x h(-1). Under the optimal condition, predicted value was 35.91%, while the experimental value was 35.07%. The experimental values agree with the predicted from the regression model with a relative error less than 5%. The main components of SPO were 9,12,15-octadecatrienoic acid, 8,11-octadecadienoic acid, octadecanoic acid and hexadecanoic acid. Most of the fatty acids were polyunsaturated fatty ones, whose quantities were obtained more than 88%. The IC50DPPH and IC50ABTS were 1.13, 3.57 g x L(-1) respectively in DPPH and ABTS assay. CONCLUSION Optimization of the extraction process by RSM of SPO is convenient and feasible. SPO has good antioxidant activity and is worth to develop for application.