The invention relates to a preparation method of a serum separation gel. The serum separation gel is prepared from the following raw materials in parts by weight: 100 parts of room temperature ion liquid, 5-10 parts of a carier and 0.02-0.1 part of a silane coupling agent. The invention further relates to the serum separation gel prepared by virtue of the preparation method. The serum separation gel can play a role of the ion liquid, the specific gravity of the separation gel can be adjusted, and the product selectivity is wide; the serum separation gel has the characteristics of being capable of resisting radiation and hydrolysis and good in stability; and the serum separation gel has the features of low toxin, environmental friendless and safety, simultaneously has a natural anti-coagulation effect, is a high-end serum separation gel and has wide market potentials.