The invention discloses a strawberry seedling substrate, and a production method and application thereof. The substrate comprises the following components: earthworm cast, peat, perlite and vermiculite, wherein the volume ratio of earthworm cast, peat, perlite to vermiculite is (1-4):(0-4):1:1; the earthworm cast is a product formed by earthworms taking cow dung. According to the strawberry seedling substrate, and the production method and application thereof, peat is replaced with the earthworm cast obtained by raising the earthworms fed with cow dung, so that resourceful utilization channels of the earthworm cast are expanded, environmental pollution is reduced, the ecological environment is protected, and the recycling and low-carbon circulation of resources are realized; adequate nutrients can be provided for strawberries in a seedling period, chlorophyll synthesis can be promoted, and the activity of peroxidase in strawberry leaves can be improved; the rooting of maternal seedlings and the growth of creeping stems are promoted, the strawberry seedling quality is improved, the rooting rate is increased, high survival rate and seedling uniformity in the seedling period are ensured, the number of strong seedlings is remarkably increased, seedling lifting labor is saved, and convenience in operation and transportation is ensured.