The invention discloses a seedling culture substrate and a preparation method of the seedling culture substrate. The seedling culture substrate comprises a substrate block (1) and a hole (4), wherein the substrate block (1) comprises garden waste compost products, vermiculite, perlite and water-retaining agents. According to any one of claim 1 to claim 8, the preparation method of the seedling culture substrate comprises the steps that garden waste is smashed to be of the length of 0.5-1cm, the C / N ratio is adjusted to be 25, and the water content is adjusted to range from 50% to 60%; organic material decomposition agents are added to the garden waste, the weight of the organic material decomposition agents accounts for 0.3% of the total weight of the garden waste, composting is carried out until decomposition is accomplished, and the garden waste compost products are obtained; the garden waste compost products, the vermiculite, the perlite and the water-retaining agents are mixed according to the volume proportion, and the pH is adjusted to range from 5.5 to 6.5; inner substrate blocks are obtained through direct block pressing production by using the mixture as raw materials, and outer substrate blocks can be obtained in a block pressing mode after slow release fertilizer and AM fungi are added in proportion. According to the seedling culture substrate and the preparation method of the seedling culture substrate, the sources of the adopted raw materials are wide, and the cost is low; plant nutrient supply is balanced; damage caused to root systems is avoided; nutrients are rich; the waste is recycled, and environment friendliness and energy saving are achieved.