The invention belongs to microbial fermentation, and particularly relates to a preparation method of a probiotic inoculum for preventing replant disease of greenhouse cucumbers. The preparation method comprises a fermentation step: inoculating a probiotic inoculum producing strain into a sterile culture medium containing a carbon source, a nitrogen source, inorganic salt and water; carrying out aerobic fermentation under the conditions that the temperature is 30-37 DEG C and the pH value is 7.2-7.6 in the fermentation process, wherein the producing strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Trichosporon behrendii, Paecilomyces lilacinus, Streptomyces microflavus, Bacillus polymyxa and Metarhizium anisopliae of the same mass; and adsorbing the final fermentation product, and immobilizing on grass carbon to obtain the probiotic inoculum. The preparation method solves the defects of replant disease, root rot, blight, nematode disease and the like in the existing greenhouse vegetable-cucumber planting technology, has the advantages of low production cost, short fermentation period and high flora density, and can effectively prevent seedling death, seedling yellowing, blight, mosaic disease, root rot and other ecological symptoms of soil. Meanwhile, the preparation method can promote the growth of roots, loosen the soil, strengthen the seedlings and degrade the residual pesticides and fertilizers, and has an effect of obviously restoring the soil ecosystem. By using the preparation method, the yield of greenhouse cucumbers can be increased by 15-20% in one season.