The invention relates to a method for preparing the transparent and conductive indium tin oxide thin film through sputtering flexible substrates at room temperature, which belongs to the technology field of electronic material; the method comprises the following steps: cleaning, charging working gas, ultraviolet irradiating, preparing a barrier layer and sputtering an indium tin oxide film; through the on-line ultraviolet irradiation, the argon oxygen ratio is (6.0 to 7.5):0.2, by adopting radio frequency sputtering or direct current sputtering, and at the room temperature, the indium tin oxide thin film can be prepared on the organic flexible substrate coated with a silicon dioxide layer in advance and taken as the diffusion barrier layer efficiently without damage. When the thickness of the indium tin oxide thin film is 700 nm, the electrical resistivity is 3.5*10-4 omega cm, the square resistance is 5 omega, the transmissibility of the visible light is 88 percent, and the film layer is even and smooth, and cannot be crimpled nor fall off. Compared with the preparation process of the traditional flexible indium tin oxide thin film, the method has the advantages that heating is not required, the process control is simple, and no damage is caused to the basal body; the method is suitable for large-area production and has good photoelectric characteristics.