The invention discloses a preparation method of humus for blueberry seedling cultivation and solves the problem that operation is quite troublesome due to the fact that in the existing blueberry cultivation process, acid increasing matter needs to be added regularly. The preparation method includes the steps that 1, water is added to grass wood materials, sandy soil and faeces, the mixture is mixed to be uniform, and no water seeps out of the mixed material when the material is held with hands, wherein the weight ratio of the grass wood materials to the sandy soil to the faeces is (10-30): 50: (10-20); 2, acetobacter pasteurianus and propionibacterium freudenreichii are added to the mixed material and stirred to be uniform; the total adding amount of the strains is at least 1*108 cuf / kg; 3, a film covers the mixed material, and fermentation is conducted for 5-8 days at the temperature of 25-28 DEG C; 4, the mixed material is cooled with cold air till the water content is 40-50%, then piled fermentation is conducted for 3-5 days at the temperature of 25-28 DEG C, and the humus is prepared. The preparation method has the advantages that the step of adding an acidic material is omitted, blueberry cultivation operation steps are simplified, and influences on growth of blueberries due to acidity difference are reduced.