The invention discloses a soilless plant cultivation substrate and a preparation method thereof. The preparation method of the cultivation substrate disclosed by the invention comprises the following steps: firstly, uniformly mixing agricultural and forestry waste, water, a fermentation catalyst, a fermentation fungicide, bamboo vinegar and/or pyroligneous liquor to carry out fermentation treatment for the first time to prepare a first fermentation substrate; and then, adding the fermentation catalyst, the water, the bamboo vinegar and/or the pyroligneous liquor into the first fermentation substrate to carry out fermentation treatment for the second time to obtain the soilless plant cultivation substrate, wherein the fermentation catalyst comprises rhamnolipid. The plant cultivation substrate prepared by the preparation method disclosed by the invention is balanced in nutrition, high in nutritional ingredient content, reasonable in structure, stable in physical property and convenient for being absorbed by plants, satisfies plant growth needs, improves survival rate for plant cultivation and ornamental effect.