Considering the biochemical conversion parameters of cultivation waste, the secondary development of product substrates and the standardized assessment after conversion of culture substrate waste, this paper takes the cultivation waste of edible fungi as the test materials and studies the microbial conversion of such waste, and then applies the converted culture substrates in the culture of cucumber seedlings. The results show that, when the cultivation waste substrate was treated through microbial fermentation, the temperature inside the substrate changed periodically. The rapeseed cake could promote the growth of the fermenting bacteria and effectively increase their metabolism; and the mixed pearlite in the waste had a positive effect in improving the porosity and water absorption of the culture substrate. The pH value decreased during the initial microbial transformation, because large amounts of organic acids were produced through the metabolism of aerobic bacteria, and then, when the temperature rose, the thermophilic microorganisms began to decompose organic acids, resulting in the continuous rise of the pH value. The higher content of substrate there was in the cultivation waste, the smaller the germination rate would be and the lower the healthy index would be, indicating that the waste substrate contained substances that inhibited seed germination. When the seedlings became adapted to the environment of the cultivation waste substrate, the nutrients in the substrate would facilitate the late-stage growth and development of seedlings.