The invention discloses horticultural long-acting and slow-release straw nutritional soil as well as a preparation method and a using method of nutritional soil substrate. In percentages by weight, the nutritional soil contains 20 percent of substrate and 80 percent of horticultural soil; the substrate contains 25 percent of perlite, 65 percent of rice straws or wheat straws, 2.0 percent of slow-release agent, 0.75 percent of calcined lime, 0.75 percent of straw well-rotten fungicide, 3.0 percent of nitrogenous fertilizer, 1.0 percent of pure phosphatic fertilizer, 1.75 percent of potassic fertilizer and 0.75 percent of microelement fertilizer; the preparation method of the substrate comprises the following steps: unbaling rice straws or wheat straws, putting under blazing sun for 3-4 daysfor sterilizing; crashing rice straws or wheat straws by a crasher into straw sections with the length of 0.8-1.2cm; and evenly mixing 25 percent of perlite, 65 percent of rice straws or wheat straws, 2.0 percent of slow-release agent, 0.75 percent of calcined lime, 0.75 percent of straw well-rotten fungicide, 3.0 percent of nitrogenous fertilizer, 1.0 percent of pure phosphatic fertilizer, 1.75percent of potassic fertilizer and 0.75 percent of microelement fertilizer to prepare the substrate, and packaging the substrate; and the using method of the nutritional soil comprises the following step: fetching 80 percent of horticultural soil and 20 percent of substrate in percentages by weight. The nutritional soil has no putrefactive odor and musty taste, synchronous straw fermentation rot and flower and plant growth as well as slowly-released nutritional ingredients, and keeps the activity and the self nutrient circulation for a long time.