The invention relates to a preparation method and application of an anti-tumor vaccine based on cell microvesicles and aims to effectively solve problems in preparation of anti-tumor vaccines high inyield, good in universality, strong in killing effect on tumor cells and capable of controlling tumors by improving tumor microenvironments, repairing an immune system and enhancing anti-tumor immuneresponse of organisms. The preparation method comprises the following steps: preparing tumor cell microvesicles loaded with immunomodulators and connecting the surfaces of the microvesicles with adjuvant-loaded liposome to stably form the anti-tumor vaccine. The prepared anti-tumor vaccine is high in yield, good in universality, strong in killing effect on tumor cells and capable of improving tumor microenvironments, repairing an immune system and enhancing anti-tumor immune response of organisms; the method is simple in process and high in efficiency; and the prepared microvesicles are derived from cells, are sufficient in quantity and wide in source, are easy for large-scale production, improve the bioavailability of adjuvants, are effectively applicable to preparation of anti-tumor vaccines based on the cell microvesicles, and can be applied to prevention and treatment of different types of tumors.