A lithium ion battery separator substrate, a preparation method and application thereof are provided. The substrate comprises a support layer and a dense layer, wherein the support layer comprises superfine main fibers, thermoplastic bonded fibers and the nanofibers, and the dense layer comprises nanofibers. The substrate has excellent high-temperature resistance performance, the substrate still has certain strength after being processed at 300° C. for 1 h, and the heat shrinkage rate is less than 5.0%; the substrate has a uniform and compact double-layer structure without a pinhole. Therefore, the requirements concerning heat resistance, porosity and strength of the substrate are met.