The present invention relates to a mold for preparing a silt soil sample based on microbial grouting. The mold comprises two templates that are in detachable connection; wherein each template comprises a bottom plate, an inner curved plate and an outer curved plate, a plurality of first circular holes are arranged on sidewall of the inner curved plate, and a plurality of second circular holes arearranged in the bottom plate, and a steel wire mesh is arranged on inner surface of the inner curved plate; a filter unit is disposed in an enclosed space on the inner curved plate above the bottom plate. The invention also provides a method for preparing the silt soil sample based on microbial grouting, the method comprises following steps: selecting a mold; culturing microorganism; measuring urease activity; putting the soil into the mold; and injecting microbial liquid and a nutrient solution along the periphery. The mold and method can be applied to silt soil with small permeability coefficient, passes the microbial liquid through the plurality of first circular holes as grouting channels to realize multi-dimensional and multipoint filling inside the soil, can effectively solve grouting uniformity without disturbing the soil, and ensure grouting effect and improving efficiency.