The invention discloses a preparation method of holozoic sea cucumber biological wine, characterized in that the method comprises the steps of: firstly soaking dry sea cucumber in warm water, filtering the sea cucumber, and leaching the aqueous solution to obtain sea cucumber soaking solution; cutting the soaked sea cucumber along the abdomen, clearing the internal organs and the limestone, flushing the sea cucumber through running water, and crushing the sea cucumber to obtain sea cucumber powder; and then, adding the sea cucumber powder in the sea cucumber soaking solution, adding neutral protease, stirring, regulating the pH to 7-8, heating to 45-48 DEG C, hydrolyzing at a constant temperature for 12-18 h, filtering, heating the filtrate to 80-90 DEG C, deactivating enzyme for 10-15 min, cooling, adding sugar substrate, common fermenting for 2-16 weeks, modulating through Chinese medicinal herb leaking solution and filtering to obtain the finished product. The prepared wine contains sea cucumber characteristic ingredients such as sea cucumber polysaccharide sulfate, sea cucumber saponin, sea cucumber amino acid and sea cucumber collagen peptide, and the wine has the functions of anti-tumor, immunity improvement, anti-coagulation, anti- thrombus, anti-radiation, anti-virus, liver protection and blood vessel protection.