Objective: The optimum conditions for the extraction of Spend Auricularia auricular substrate by supercritical CO₂ fluid were studied, and the chemical composition of the extract was analyzed. Methods: Taking the extraction rate as the indicator, orthogonal test of L9 (3⁴) with four factors and three levels was carried out to determine the best conditions on the basis of single factor experiment. The extraction was analyzed with gas chromatograph/mass spectrometer. Results: The results showed that the optimal extraction conditions were 30 MPa, 45 ℃, 60 mesh, 400 mL of the entrainer volume and the extraction rate was 3.25% under these conditions. The chemical composition of extraction can be classified into alcohols, ethers, aldehydes, ketones, organic acids, esters, terpenoids, steroids. The three most abundant compounds were methyl linoleate (5.56%), 17β(H),21β(H)-hop-22(29)-EN-3-one(5.26%),3,5-cholestanedien-7-one(4.05%),respectively. Conclusion: Extraction and separation of fine chemicals from spend auricularia auricular substrate is a new idea of clean and efficient utilization of biomass.