As one of the most appealing edible mushrooms, the quality of Flammulina velutipes can be affected by the cultivation substrate, which has an impact on the mushroom umami taste and nutrients. In our study, the effect of mushroom root fermentation broth (MRFB) based substrate on umami taste and nutrients of F. velutipes were evaluated; four proportions of MRFB were conducted (CK 0%, E1 6.7%, E2 13.4%, E3 20.1%). Results indicated that MRFB was an effective nutrient supplement for mushroom cultivation substrate. E2 and E3 showed higher crude fiber, crude fat, and soluble protein. Compared to CK, the content of monosodium glutamate-like (MSG-like) amino acids, essential amino acids, total amino acids, and total 5’-nucleotides in E2 and E3 were higher. The equivalent umami concentration (EUC) values in E1, E2 and E3 were 1.70, 2.43 and 1.56 times that of CK, respectively. Higher umami, saltness and sweetness taste values were found in E2. Thereby, it signified that better umami and richer nutrients were achieved by using substrate with proper volume of MRFB, especially, E2 with 13.4% of MRFB. Overall, better mushroom quality could be derived from the application of MRFB in cultivation. MRFB was a favorable nutrient supplement for F. velutipes cultivation substrate.