The invention relates to a high-barrier transparent flexible display material and a preparation method thereof. The preparation method comprises the following steps: firstly heating an anionic two-dimensional layered material and an organic intercalation agent in a solvent, then performing a stripping treatment in the solvent, mixing the material with a polymerization monomer of polyimide and performing in-situ polymerization, and finally performing heat treatment to obtain the flexible display substrate material. The material obtained by the preparation method of the present invention has high water-oxygen barrier property, excellent thermal stability, low thermal expansion coefficient, and high transparency and the like, and is the flexible material excellent in comprehensive performance.