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In this study, GC-MS/SIM and HPLC methods were successfully applied for determination of total ergosterol and at-soluble vitamins in the spore lipids of G.lucidum, coupled with supercritical fluid extract (SFE) in an off-line mode.At first, the operational conditions were designed on 30MPa of extract pressure and 40℃of extract temperature, 25L/h of CO2 flux, 2.0h of extract time.And the spore lipids was collected from separator I (at 8 MPa, 45℃) with averaged yield of 24.16%(w/w), while the extract rate was 95.08% (R (%) =[1-A1 / A0] × 100; A0=content of ctude fat in the spores ; Al=content of crude fat in the remains after SFE, by Soxhlet-extract method).