The invention relates to a preparation method of medicinal fungal mycelium glycoconjugate, and especially relates to an ultrasound assisted method for preparing glycoconjugate from medicinal fungal mycelia through utilizing a high pressure homogenizer. The method comprises the following steps: pre-crushing mycelia obtained after the submerged fermentation of a liquid by a tissue crusher, adding the pre-crushed mycelia to a high pressure homogenizer, crushing for 10min, adding deionized water having a pH value of 7 and an amount being 10-30 times the amount of mycelia, and homogenizing under a pressure of 60-200MPa 1-3 times; placing the obtained homogeneous liquid in an ultrasonic wave extracting machine, and extracting for 15-20min, wherein the ultrasonic power is 80-120W, the ultrasonic temperature is 30-40DEG C, the work time is 2s, the interval time is 3s, and the ultrasonic frequency is 2; and centrifuging, removing a precipitate, adding anhydrous ethanol having a volume 3 times the obtained supernatant to the supernatant for precipitation, filtering the obtained precipitate, and drying to obtain the fungal mycelium glycoconjugate. The contents of total sugar, reducing sugar, polysaccharides and peptides in the obtained glycoconjugate are 30-60%, 0.2-3%, 30-57% and 20-30% respectively. The glycoconjugate yield is 1.0-3.0g/L. The method has the advantages of short extraction time, high extraction efficiency, low extraction cost, small destroy to active components, convenient operation, high automation degree and the like.