Defatted soy flour is a potential source of food protein, amino acids, ash and isoflavones. The supercritical carbon dioxide (SC-CO2) and a traditional organic solvent extraction methods were used to remove fat from soy flour, and the quality characteristics of a control soy flour (CSF), defatted soy flour by SC-CO2 (DSFSC-CO2) and defatted soy flour by an organic solvent (DSF-OS) were compared. The SC-CO2 process was carried out at a constant temperature of 45 °C, and a pressure of 40 MPa for 3 h with a CO2 flow rate of 30 g/min. The DSFSC-CO2 had significantly higher protein, ash, and amino acids content than CSF and DSF-OS. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that CSF and DSFSC-CO2 had protein bands of similar intensity and area that indicated no denaturation of protein, whereas DSF-OS showed diffuse bands or no bands due to protein denaturation. In addition to higher nutritional value and protein contents, DSFSC-CO2 showed superior functional properties in terms of total soluble solids content, water and oil absorption, emulsifying and foaming capacity. The SC-CO2 method offers a nutritionally and environmentally friendly alternative extraction processing approach for the removal of oil from high-protein food sources. It has a great potential for producing high-protein fat-free, and low-calorie content diet than the traditional organic solvent extraction method.