Objective: Measure and compare the content of free amino acid in Hypsizigus marmoreus, Pleurotus eryngii and Flammulina velutipes. Method: The free amino acids in Hypsizigus marmoreus, Pleurotus eryngii and Flammulina velutipes were pretreated with phenyl isothiocyanate and the content of samples was determined by HPLC. Results: The content of total free amino acid in Hypsizigus marmoreus, Pleurotus eryngii and Flammulina velutipes was respectively 5.3185%, 4.5863% and 4.7222%. The content of essential amino acids was respectively 2.6701%, 2.0407% and 1.5963%. The content of children's essential amino acids was respectively 0.2327%, 0.1827% and 0.6099%. The content of medicinal amino acids was respectively 3.1061%, 2.7769% and 3.3132%. The content of the delicious amino acid was respectively 1.0741%, 1.0285% and 1.4690%. Conclusion: Hypsizigus marmoreus is rich in nutritional value and medicinal value, it is worth to developing and using.