[Objective] To eliminate virus contamination in Flammulina velutipes, we assessed five detoxification methods. [Methods] F. velutipes F4889 was used to isolate 2.0 kb dsRNA, and the virus was identified as F. velutipes browning virus (FvBV) by RT-PCR. Five virus-eliminating methods, i.e. hyphal tips isolation virus elimination (HTIVE), primordium tissue isolation virus elimination (PTIVE), protoplast monokaryotization virus elimination (PMVE), sexual reproduction virus elimination (SRVE) and nuclei migration virus elimination (NMVE) were applied to prepare virus-free strains. The effect of virus-elimination was detected and identified through dsRNA technique and RT-PCR. [Results] Only one virus-free strain was obtained by HTIVE method. Strains of PTIVE method remained the virus. PMVE method achieved three monokaryon strains and two protoplast monokaryon hybridization virus-free strains. Twenty-three sporulated monocaryotic strains and 8 single spore hybridization strains were virus-free after using SRVE method. NMVE method acquired 5 virus-free strains. Hyphal growth rate, biomass and laccase activity of virus-free strains by PMVE, SRVE and NMVE methods were obviously superior to the original strain and strains by HTIVE and PTIVE methods. [Conclusion] The detoxification effect of PMVE, SRVE and NMVE methods was apparently better than others. These three methods completely eliminated FvBV and had higher virus-free rate. Moreover, mycelia growth rate, biomass and laccase activity of virus-free strains significantly improved.