The invention discloses a cell freezing medium as well as a preparation method and application thereof. Comprising the following components: an IMDM basal culture medium, DMSO, 4-hydroxyethyl piperazine ethanesulfonic acid, pyridoxal hydrochloride, inositol, putrescine, serotonin, mercaptoethanol, arachidonic acid, cholesterol, catechin, bile alkali chloride, calcium pantothenate, nicotinamide, trehalose, polyoxypropylene polyoxyethylene, Tween 80, albumin, transferrin, fibronectin, fisetin, piperlongumine, tocopheryl acetate and linoleic acid. Linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid; the composition further comprises amino acids, hormones, inhibitors, cell factors, inorganic compounds and vitamins. The cell cryopreservation liquid provided by the invention is used for cryopreserving mesenchymal stem cells, so that the human umbilical cord mesenchymal stem cells have high survival rate after cryopreservation and resuscitation, and meanwhile, a better differentiation capability is kept; and the cell survival rate and proliferation activity can be better maintained.