To study composition and content of protein amino acids (PAA) and nonprotein amino acids (NPAA) in Letinous edodes and Flammulina velutipes, and to evaluation their nutrition and flavor, the composition and content of PAA and NPAA in Letinous edodes and Flammulina velutipes were determined by OPA-FMOC precolumn derivatization and HPLC. The results showed that Letinous edodes contains 15 kinds of PAA with the content of 10.99 mg/g, contains 2 kinds of NPAA with a content of 2.37 mg/g. The content of essential amino acids (EAA) is 4.39 mg/g and the content of flavor amino acid (FAA) is 5.40 mg/g. There are 14 kinds of PAA in the Flammulina velutipes with content of 16.79 mg/g and 2 kinds of NPAA with the content of 3.99 mg/g. The content of the EAA is 5.98 mg/g and FAA is 9.96 mg/g. The method is accurate and feasible. It can be used for the determination of PAA and NPAA in the fungi. There are more kinds of PAA in Letinous edodes than that in Flammulina velutipes, EAA in Letinous edodes was more than that in Flammulina velutipes, and PAA and NPAA contents in Flammulina velutipes were higher than those in letinous edodes. Flammulina velutipes occupy high nutritional value and delicious taste, while Letinous edodes possess a balance nutritional value. The results in this work were helpful for the re-development of Letinous edodes and Flammulina velutipes.