The invention provides an extraction method of an ergosterol monomer compound in Armillarialuteo-virens. The method includes: taking an Armillarialuteo-virens fruit body as the raw material, grinding the raw material into powder, then conducting extraction with ethyl acetate, and carrying out concentration into paste; conducting dissolving with a n-hexane-ethanol solvent; conducting one-dimensional liquid chromatography separation: employing a Silica chromatographic column, adopting a binary mobile phase with n-hexane as the phase A and ethanol as the phase B, performing gradient elution with phase B at a concentration ranging from 0% to 75%, and collecting the 8-11min eluent; performing dissolving with an ethanol-n-hexane solvent; carrying out two-dimensional liquid chromatography separation: adopting a Silica chromatographic column, employing a binary mobile phase with n-hexane as the phase A and ethanol as the phase B, performing isocratic elution with phase B at a concentration of 2-6%, collecting the 12-15min eluent, and performing evaporation concentration to obtain ergosterol. The separation method has the advantages of stability, high repeatability, large preparation capacity, simple operation and time saving, low cost, and high ergosterol extract rate. The target object has an inhibiting effect on lung cancer A549 cells and liver cancer Hep-G2 cells, and has anticancer drug development potential.