The present invention relates to the field of arbuscular mycorrhiza fungi test apparatus, particularly to a double room hypha test apparatus and a double room hypha test method. The double room hypha test apparatus comprises a plant room, a filtration screen, an air separation layer and a hypha room from top to bottom, wherein the upper portion of the plant room is provided with an opening, the edge side of the plant room is sealed, the filtration screen and the air separation layer are arranged on the bottom portion of the plant room, the upper portion of the hypha room is provided with an opening, the edge side of the hypha room is provided with a ventilation gas permeation hole, the bottom portion of the hypha room is the completely-closed structure, and the plant room and the hypha room are connected through a bolt. According to the present invention, with the air separation layer method, the root system and the hypha can be well separated; and the upper compartment and the lower compartment are adopted, such that the gravity effect can be well utilized, the hypha growth is easily achieved, the nutrient diffusion influence between the two rooms due to the mass flow can be reduced, and the good test effect is achieved.