A method for preparing a probiotic preparation based on a W1/O/W2 double emulsion structure includes steps of: using a solution containing probiotics or a mixed solution of probiotics and a probiotic protective agent as an internal water phase W1; dissolving an emulsifier in a lipid phase O, and mixing the lipid phase O with the internal water phase W1; then preparing a primary emulsion W1/O by stirring and low-energy emulsification or high-energy emulsification; and using an emulsifier-contained solution as an external water phase W2, and adding the external water phase W2 to the primary emulsion W1/O; then preparing a W1/O/W2 double emulsion by stirring and the low-energy emulsification or the high-energy emulsification, thereby obtaining the probiotic preparation. The double emulsion system is used to embed the probiotics, which can keep the probiotics active during low-temperature storage and freezing and thawing.