The invention provides novel non-sintered biological stuffing rapid in biofilm formation and a preparation method thereof, relating to biological stuffing and preparation methods thereof and solving the technical problems that the existing biological stuffing has long biofilm formation time and poor effluent quality in water treatment processes and preparation processes are high in energy consumption and pollute the environment. The biological stuffing is prepared from sepiolite, medical stone, cement, polyvinyl alcohol, sodium alginate, hydrogen peroxide, ferric chloride, manganese sulfate, cobalt chloride and agar. The method comprises the following steps: I. heating and dissolving polyvinyl alcohol and sodium alginate, adding hydrogen peroxide after cooling, and mixing the components uniformly, thus obtaining a liquid mixture; II. mixing the liquid mixture with other solid components, granulating the mixture, putting the granules at normal temperature, drying and sieving the granules, curing the granules at high pressure and low temperature, steam-curing the granules at constant temperature, and cooling the granules at normal temperature, thus obtaining the biological stuffing. Compared with the common ceramsite, the biological stuffing has short biofilm formation time, good effluent quality and coarse surface and is beneficial for microorganism growth. The biological stuffing is applied to the fields of environmental applied materials and sewage treatment.