The invention provides a preparation method of a high-purity aluminum oxide ceramic substrate, which comprises the following steps: S1, putting 99-99.5 parts by weight of high-purity aluminum oxide powder and 0.5-1 part by weight of a sintering aid into a ball mill, adding a water-soluble dispersing agent by using water as a solvent, and grinding and mixing to obtain slurry; S2, filling the slurryinto a slurry barrel after vacuum defoaming, adding a catalyst and an initiator, uniformly stirring, connecting an air source, opening the air source to apply pressure to the slurry to control the slurry to be injected into a mold from a slurry injection port in the bottom of the mold through a communicating pipe, enabling the slurry to flow in the mold from bottom to top until the mold is filledwith the slurry, curing and demolding to obtain a green body; S3, drying the green body, softening, cutting the green body into green body slices, and drying; and S4, sintering the green body slices,and flattening to obtain the substrate. The substrate is low in sintering temperature, wide in sintering range and controllable in sintering contractibility.\ The air source is used for applying pressure to slurry, so that the slurry is injected into the mold from bottom to top from the bottom of the mold, and the problems of large blank density difference and large molding defects caused by deposition of materials with high specific gravity in the slurry to the bottom of the mold are solved.