The invention discloses a device and a method for preparing a microkey abhesion experiment sample at high throughput. The device comprises a mould, a monofilament positioning fork and a monofilament positioning fork fixing device; the mould is provided with a plurality of round holes and is formed by processing a series of round holes in a rectangular plate; the monofilament positioning fork fixing device comprises a vertical plate and two horizontal plates, the two horizontal plates are fixed at the upper part and the lower part of the vertical plate to form a C-shaped structure, and a monofilament positioning fork fixing groove is formed in the middle part of the vertical plate; monofilament positioning fine seams are formed in one end of the monofilament positioning fork, the number ofthe monofilament positioning fine seams and the number of the round holes are the same, and the other end of the monofilament positioning fork is inserted into the monofilament positioning fork fixinggroove to be fixed; and a threaded hole is formed in the top part of the monofilament positioning fork fixing device, and a bolt is used to fasten the monofilament positioning fork. The time for preparing the microkey abhesion experiment sample can be shortened, the material and the time are saved, and up to 50 samples can be prepared at one time.