A wild Flammulina velutipes strain was used as the test material to investigate screening methods for Flammulina velutipes strains suitable for straw cultivation in test tubes. Tissue and spore isolation of cultivated Flammulina velutipes were performed, and the cultivated strains were tested. The results showed that Flammulina velutipes can be grown successfully in closed test tubes with straw as the cultivation medium. The subsequent tissue isolation and spore isolation are convenient and successful and can be used for preliminary screening of strains for straw cultivation.