The invention discloses a mulberry layer cake. The cake includes a cake body and a filling. The cake body is prepared from, by weight, 450-550 parts of low-gluten wheat flour, 17-25 parts of milk powder, 8-9 parts of baking powder, 230-240 parts of granulated sugar, 265-275 parts of fresh eggs, 2.5-5.5 parts of edible salt, 116-126 parts of edible oil, 62-72 parts of sorbitol, 1-2.5 parts of sodium bicarbonate, 4-8 parts of cake oil and 80-150 parts of water. The filling is prepared from, by weight, 40-50 parts of mulberry pulp, 40-50 parts of mulberry jam and 800-900 parts of lotus paste. Themulberry layer cake has the advantages of being low in fat content, unlikely to go bad, endurable to long-term preservation, abundant in nutrient and capable of achieving the functions of relaxing bowels, nourishing skin and the like. The invention also discloses a preparation method of the mulberry layer cake. The preparation method is simple in flow, convenient to operate and low in cost.