The invention provides an anti-freezing spacer fluid and a preparation method and the application of the anti-freezing spacer fluid. The anti-freezing spacer fluid comprises a compound anti-freezing solution and a cosolvent, wherein the mass ratio of the compound anti-freezing solution to the cosolvent is 100 to 1-2, the compound anti-freezing solution comprises three anti-freezing agents and water, the three anti-freezing agents are mutually soluble with water, and the mass ratio of the three anti-freezing agents to the water is 40-100 to 10-60. The preparation steps are simple, the used materials are simple and economical, the operation process is convenient, no hydrate or obstruction is generated when the anti-freezing spacer fluid is used in the synergistic transformation of liquid carbon dioxide dry fracturing and hydrofracturing of a low-permeability gas reservoir, the spacer liquid basically does not change the viscosity of a fracturing fluid, shows better compatibility, does not freeze at the temperature of minus 51.5 DEG C, keeps low viscosity and low frictional resistance, is excellent in flowability, can still resist the low temperature of minus 30 DEG C after being mixed with the water-base fracturing fluid, has an obvious anti-freezing effect, and is of great practical significance.