The invention provides a lentiviral vector cold storage protection liquid. The lentiviral vector cold storage protection liquid comprises the following components by volume fraction: 88-97% of a tris-HCL buffer solution containing NaCl, 1-10% of a B27 serum-free additive, 1-6% of a CD-Lipid concentrated solution and 1-8% of a human serum albumin (HSA) solution, wherein in the tris-HCL buffer solution containing NaCl, the molar concentration of tris-HCl is 0.8-20 mM, and the molar concentration of NaCl is 0.08-0.15 M; and the pH of the tris-HCL buffer solution containing NaCl is 7.0-8.0. The cold storage protection liquid can store a lentiviral vector at a relatively low temperature for a long time without influencing the activity, can also be repeatedly frozen and thawed in the use process, and can still maintain the activity, meeting the use demands, of the lentiviral vector after being exposed at the body temperature of an organism or the room temperature. The invention further provides a preparation method and application of the cold storage protection liquid.