The invention belongs to the technical field of the drug screening chip, and specifically relates to a high-flux microplate drug screening chip based on electrochemical transistor, and a preparation method thereof. A to-be-solved technical problem is to provide a high-flux microplate drug screening chip based on an electrochemical transistor, and a preparation method thereof. A technical scheme for solving the technical problem is as follows: a chip comprises a three-layer structure orderly including a PCB board, a through hole layer and a substrate from a top layer to a bottom layer; multiplesections of metal wires with same structure, multiple PCB board rectangle bonding pads and multiple PCB board round bonding pads are welded on the PCB board; the metal wires are inserted into the corresponding PCB board round bonding pads and fixed by using soldering time; the PCB board round bonding pads are connected with the PCB rectangle bonding pads through conductors on the PCN board surfaces; multiple round through holes are arranged on the through hole layer; multiple substrates pad, a microelectrode array and multiple conductors are arranged on the substrate; the microelectrode arrayis connected with the substrate pad through the conductors. The preparation method of the high-flux microplate drug screening chip disclosed by the invention is applied to the manufacturing of the high-flux microplate drug screening chip.