The invention provides a high-safety non-inactivated virus preservation solution and a preparation method thereof. The preservation solution includes hanks base solution, antibiotic medicament, protein protectant, cryoprotectant and nutrient, and the protein protectant includes bovine Serum albumin, mannitol, glycerin, butylene glycol, sodium chloride and sodium benzoate, described cryoprotectant comprises sodium alginate, polyethylene glycol, glucose, and described nutrient comprises asparagine, glutamine, threonine, aspartic acid, valine, leucine, isoleucine, said antibiotic agent includes gentamicin, amclocillin sodium and amikacin. Inhibit the influence of bacteria and fungi on viruses by antibiotics, protect the protein shell of viruses by protein protectants, stabilize the protection solution at low temperatures by cryoprotectants, and enable viruses to survive in the protection solution by nutrients .