High ergosterol-producing yeast strains were constructed by primary screening isolation of haploid mutagenesis and protoplasts fusion. The fermentation conditions of fused strain YEF-21 were studied. Under the optimal conditions, i.e., the medium for fermentation consisted of 8% glucose 1% polypeptone 1% yeast extract; initial pH nature; 60 mL broth/250 mL flask; inoculum volume 10%; fermentation time 30 hours at 28 degrees C and 200 r/min; the comprehensive value of biomass and ergosterol content of YEF-21 is 1.54 and 1.55 times that of the parent strains YE227 and YE180, respectively. The fused strain YEF-21 is stable enough genetically according to the analysis of genetic stability, and it is a high ergosterol-producing strain that has prospect for application.