The invention discloses a liquid culture substrate for rose seedlings and a preparation method of the liquid culture substrate. The substrate is prepared from raw materials (in parts) by weight as follows: an alga mixed solution, potassium ferrate, aluminum sulfate, sodium molybdate, zinc acetate, bone meal, alfalfa, soybean meal, strain powder, turf, seabuckthorn fruits, phosphorus pentoxide, expanded perlite, mushroom residues, urea, ammonium hydrogen carbonate, sheep dung balls and a proper amount of water, the rose seedlings are cultured in a soil and stone powder mixed substrate, on one hand, nutrients cannot be well controlled, conditions of uneven growth and maldevelopment of partial seedlings can be caused easily, soil carries germs and eggs easily, roses in a large area are infected once infection occurs, the substrate is in the liquid state, required nutrients can be added at any time according to the growth vigor, a large quantity of inorganic fermented matters are added, resources are saved, and growth of the roses in the juvenile stage is facilitated.