The invention discloses a preparation method of a bottled armillaria mellea strain. The method comprises the following steps of: mixing: weighing culture materials according to a culture medium formula and mixing uniformly in dry state; B, bottling: filling the uniformly-mixed dry culture materials into strain bottles; C, basketing and filling water: putting the filled strain bottles into a disinfection transition basket, immersing the entire basket into a water pool, and filling water; D, pre-sterilizing: pre-sterilizing the water-filled strain bottles; E, discharging: discharging the entire basket of pre-sterilized strain bottles out of a sterilizing stove; F, sealing: plugging and sealing the strain bottles with common strain sealing articles such as bottle plugs, bottle caps, cotton plugs and kraft paper; G, secondary sterilizing: transporting the sealed strain bottles into the sterilization stove once again for normal-pressure or high-pressure sterilization; and H, cooling and inoculating, wherein the strain bottles are required to be kept in upright state in the entire production process. Due to the adoption of the method, the bottling speed during armillaria mellea strain production can be increased, the sealing articles are prevented from being polluted, strain microbial contamination is reduced, and factory production is facilitated.