Diets rich in flavonoid-containing foods are sometimes associated with cancer, neurodegenerative and cardiovascular disease prevention. β-glucan, one of the most important bioactive compounds in medicinal mushrooms, is used to boost the immune system, diabetes and cancer. In this study, we aimed to determine the content of two flavonoid compounds (naringenin and naringin) and β-glucan in Flammulina velutipes and Flammulina velutipe-gold strain. The naringenin, naringin, and β-glucan components from the Flammulina velutipes and Flammulina velutipe-gold strain were measured by the HPLC analysis. As a result, the contents of naringenin, naringin, and β-glucan were found to be much higher in Flammulina velutipe-gold strain than Flammulina velutipe. From the above results, we may suggest that Flammulina velutipe-gold strain might have useful as a material for functional food and pharmaceutics fir the pathological process of various human diseases.