The invention relates to a preparation method of a biological microelectrode array based on a flexible substrate, which adopts secondary photolithography and electroforming technology, and sequentially coats polydimethylsiloxane sacrificial layer and polyimide acid on the substrate surface, Thermal curing forms a polyimide flexible substrate, and prepares metal circuits on the flexible substrate by sputtering, photolithography and electroforming processes, then etches the metal base film, coats polyimide acid, photolithography and etch polyimide Imino acid, electroformed metal electrodes, thermally cured to form polyimide, and the prepared biomicroelectrode array based on polyimide flexible substrate was peeled off from polydimethylsiloxane. The electrodes are located at both ends of the metal circuit, and the electrode array is arranged according to the application requirements. One end of the electrode is connected to the biological tissue, and the other end is connected to the stimulator or measuring instrument. The metal circuit at the bottom of the electrode connects the electrodes on both sides. The biological microelectrode array manufactured by the invention has the advantages of high yield, controllable electrode position, small impedance, biocompatibility and the like.