The invention discloses an agarose gel substrate and a preparation method thereof. The preparation method comprises the steps that water, agar powder and inorganic salts are mixed, the first mixture is heated and mixed to boiling point, and the agar powder and inorganic salts are dissolved, and a first solution is obtained; the pH value of the first solution is regulated, the first solution is poured in a container, after the bubbles are removed, the solution is cooled at room temperature to solid gel like, and a first nutrition layer is obtained; water and agar powder are mixed, the second mixture is heated and mixed to agar powder and dissolved, and a second solution is obtained; the pH-value of the second solution is adjusted; the second solution is cooled at room temperature and poured on the first nutrition layer, after the bubbles are removed, the solution is continued being cooled to a solid gel shape at room temperature, and a second nutrition layer is obtained. The obtained agarose gel substrate is used for the soilless culture of plants, with the substrate being not splashing, the users can view through the substrate the cultivated plant roots, the watering cycle is flexible without having to constantly add or replace nutrients, and user experience is improved, and the agarose gel substrate has the characteristics of being repeatedly reusable, nontoxic and low in cost.