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The present application relates to the technical field of biological medicine, in particular to an anti-tumor grifola frondosa water-soluble protein complex preparation and a preparation method thereof.The preparation raw materials of the complex preparation include the following components in parts by weight: grifola frondosa water-soluble protein 2-3 parts, fermentation preparation 0.8-1.2 parts, rosemary acid 0.1-0.2 parts.