The invention relates to a soilless seedling culturing substrate for resisting diseases and root nematode of plants and a production method thereof. In the invention, organic wastes such as straws are taken as raw materials, inoculated with zymogen according to certain proportion, subjected to moisture content regulation, evenly mixed and stacked for primary fermentation; when the temperature of a pile reaches 60 DEG C, the pile is turned; the pile is turned and oxygen and moisture content are supplemented every seven to ten days afterwards; after the pile is turned for three times, when the temperature is lower than 35 DEG C, the pile is inoculated with a bio-antagonist for secondary fermentation, turned every three to four days and kept at a temperature of between 35 and 40 DEG C; and after 15 to 20 days, the seedling culturing substrate containing the 108/gram bio-antagonist is obtained. The seedling culturing substrate has chemiotaxis for interfering pathogenicbacteria and nematode, can reduce infestation chance of the pathogenicbacteria and nematode on the root surface, and has double effects of resisting phytophthora disease and root nematode disease through the secondary fermentation and microorganism activation technology.