The invention relates to the technical field of soilless culture, and particularly provides a soilless culture substrate with moisture and fertilizer conservation capacity. The soilless culture substrate is characterized by being prepared from the following various raw materials in parts by weight: 30 to 50 parts of biological organic fertilizer, 5 to 10 parts of turf, 10 to 15 parts of fine sand, 5 to 8 parts of coarse sand, 1 to 5 parts of perlite, 2 to 5 parts of calcium magnesium potash powder, 8 to 20 parts of weathered coal, 1 to 3 parts of powdered rock phosphate, 1 to 7 parts of medical stone, 22 to 33 parts of vermiculite, 62 to 73 parts of plant residues, 0.6 to 2 parts of a water-retaining agent and 6 to 10 parts of enzyme microorganism biological organic fertilizer. A preparing method of the soilless culture substrate with moisture and fertilizer conservation capacity comprises a material preparation step, a rotting step and a mixing step. The soilless culture substrate with moisture and fertilizer conservation capacity is obviously high in air permeability, buffering property, water conservation capacity and fertilizer conservation capacity, and has good other physical and chemical indexes; the preparing method of the soilless culture substrate with moisture and fertilizer conservation capacity is simple, convenient and low in labor cost.