The invention discloses a southern yew container seedling substrate, which is formed by adding decomposed vinegar grains to fresh vinegar grains and sawdust, adding urea to the sawdust, performing primary fermentation, adding perlite after mixing, and performing secondary fermentation. The bulk density of the prepared matrix is 0.40±0.02g / cm 3 , the total porosity is 62.8±2.2%, the water-holding porosity is 38.8±1.1%, and the air porosity is 24.0±1.3%. A preparation method thereof is also disclosed. The matrix of the present invention is mainly prepared from vinegar grains and sawdust organic waste, and the preparation process is simple and easy to operate; the raw materials involved are mainly fermented organic waste products, and the amount of bacterial agent used is greatly reduced, which reduces the production cost , and at the same time realize the comprehensive utilization of waste resources and reduce environmental pollution. The prepared matrix not only has a good seedling raising effect, but also can replace the conventional peat matrix, with low cost and broad market prospects; the matrix prepared according to the preparation method can significantly increase the plant height, ground diameter and total branch number of the southern yew container seedlings, and improve the leaf quality. Chlorophyll content.