The invention discloses a preserving method of large ball-cover mushroom strain comprising a) configuring directing domestication plating medium, b) preparing non-freezing, non-negative pressure mycelium, c) freezing and drying the mycelium in vacuum: putting the ampoule pipe into refrigerating drier to freeze to -40 DEG C at the speed of 2 DEG C per minute, pre-freezing in constant temperature for 1.5 hours and then opening the vacuum pump, after the vacuum degree achieving 22.5 Pa within 15 minutes, the temperature of the isolation plate rising 5 DEG C and the vacuum degree reducing 5Pa per 30 minutes until the temperature of the isolation plate rising to 24 DEG C and the vacuum degree reducing to 0.8 to.