The invention relates to a preparation method of a tricholoma matsutake mycelium fermented vinegar beverage. In order to solve the problems that in the prior art, tricholoma matsutake sporophore is used as a raw material, so that the use form is single, the cost is high, and the growth period is long, the invention provides the preparation method of the tricholoma matsutake mycelium fermented vinegar beverage. According to the preparation method, tricholoma matsutake mycelia are used as raw materials, alcohol fermentation and acetic acid fermentation are performed to obtain mycelium original vinegar, wherein a response surface optimization analysis method is used to analyze the fermentation process, so that optimum parameters are obtained, and finally, with preserved fruit syrup and honey as blending raw materials, blending is conducted by adopting an orthogonal test method. The conditions of the alcohol fermentation are determined: fermentation temperature is 28.5 DEG C, fermentation time is 6 d, pH is 5, and inoculation amount is 1.4%; the conditions of the acetic acid fermentation are also determined: fermentation temperature is 28.0 DEG C, the rotation rate of a shaker is 146 r/min, and inoculation amount is 10%; and the tricholoma matsutake mycelium fermented vinegar beverage is prepared from 10% of tricholoma matsutake original vinegar, 8% of the preserved fruit syrup, 5% of the honey and 77% of water. The preparation method can be applied to the field of development of new mycelium health-care products.